HK1159695A

HK1159695A - Method for labelling a product using a plurality of polynucleotides, method for identifying the labelling and labelled product

Info

Publication number: HK1159695A
Application number: HK12100061.0A
Authority: HK
Inventors: 亚历山大‧雅各布; 卡洛斯‧克梅格尼‧卡埃莫; 西尔万‧劳瑞克; 斯特凡‧蒙特奥斯; 尼古拉斯‧德拉考特
Original assignee: 生物量子公司
Priority date: 2008-04-14
Filing date: 2009-04-10
Publication date: 2012-08-03

Description

Method for labeling product using multiple polynucleotides, method for identifying said label, and labeled product

Technical Field

The present invention relates to a method for marking a product, a method for authenticating said marking and a product marked by the method of the invention. The labels used in the present invention are based on single-stranded nucleic acids.

The invention makes it possible to distinguish between genuine and counterfeit items. The invention makes it possible in particular to mark genuine articles so that they can be tracked and authenticated.

Counterfeiting or illegal copying of articles, particularly high value-added products or high-end products, causes serious economic losses not only to companies, but also to employment, food safety and even social life. This is why manufacturers strive to combat this mishap by developing new marking and authentication techniques for their products to track and destroy counterfeit products.

The reference ([ x ]) between brackets refers to the list of references at the end of the example.

Background

One of the methods commonly used to detect and identify genuine products is the "bulk" labeling of the product by introducing a chemical or compound to the product that can be identified in a single way.

This type of marking must have specific properties: the marking must be done in a manner that is transparent to the end user of the product, should not change the physico-chemical properties of the product and should not be harmful to the end user of the product. In practice, the mark should also be undetectable and/or not be counterfeited, so that it does not itself be counterfeited at the same time as the product.

At present, particularly for very large amounts of chemicals such as fragrances, cosmetic creams, etc., which are known to be critical for markers, or for materials such as leather, textiles, etc., no reliable technology is available. Today there is no marking technology that makes a true decryption difficult for counterfeiters.

There is therefore a real need for a marking and a method of marking which overcome these drawbacks, disadvantages and obstacles of the prior art and which makes it possible to effectively combat counterfeit products, in particular counterfeit high-end products.

Disclosure of Invention

In particular, the object of the present invention is to propose a marking scheme that satisfies the above-mentioned needs and solves the problems of the prior art.

The invention relates inter alia to a method of product labelling comprising the step of adding a plurality of single stranded polynucleotides on or in said product, said plurality of polynucleotides comprising:

-at least one target polynucleotide comprising a single stranded polynucleotide having a predetermined length and sequence, and

a decoy polynucleotide (decoy polynucleotide) of the same or different predetermined length and of the same or different predetermined sequence, said decoy polynucleotide being of the same or different length as said at least one target polynucleotide and the sequence of said decoy polynucleotide being different from the sequence of said at least one target polynucleotide,

wherein each of the target polynucleotide and the decoy polynucleotide does not hybridize to any other polynucleotide of the plurality of polynucleotides.

In a particular embodiment of the method of the invention, the plurality of polynucleotides further comprises at least one identification polynucleotide consisting of a single-stranded polynucleotide having a predetermined length and sequence and for identifying the nature and sequence of the at least one target polynucleotide, wherein each of the identification polynucleotides does not hybridize to any other polynucleotide of the plurality of polynucleotides.

With respect to the method of the present invention as defined above, and with respect to the specific embodiments of the method of the present invention, the following detailed description should be considered.

Thus, the marker of the invention consists of said plurality of polynucleotides as defined in the specification. These polynucleotides are single stranded polynucleotides. In the present specification, the plurality of polynucleotides is also referred to as "marker".

The invention also relates to a marked product obtainable by the method of the invention, and to a method for detecting the marking of such a product.

In particular, the invention relates to the determination of markers for carrying out the method of the invention, the manufacture of these markers, the marking of products and techniques for detecting markers in marked products. The invention makes it possible to distinguish between counterfeits and genuineness and even to identify misuse of distribution channels and unauthorized parallel channels.

The polynucleotides in the plurality of polynucleotides may be of several types: they may be polymers of ribonucleotides or single-stranded Ribonucleotides (RNA) or deoxyribonucleotides or single-stranded deoxyribonucleic acids (DNA) or combinations of these.

In the present invention, "plural polynucleotides" refers to a group of target polynucleotides, decoy polynucleotides, and identification polynucleotides as necessary. Preferably, according to the present invention, several target polynucleotides, several decoy polynucleotides, and if necessary, several identification polynucleotides are used. For example, but not limited to, 1 to 100 target polynucleotides, such as 1 to 50, for example 5 to 50, can be used. In addition, for example, but not limited to, 2 to 100 decoy polynucleotides, such as 2 to 50, can be used. For example, but not limited to, if necessary, 1 to 100 kinds of recognition polynucleotides, for example, 1 to 50 kinds, may be used. The number of polynucleotides selected depends on the complexity of the label required according to the invention.

In the present invention, the "target polynucleotide" refers to a polynucleotide whose sequence has been determined and which is constructed to constitute a reference sequence, the target polynucleotide being used to label the product of the present invention and then specifically searched for in the product, thereby authenticating the product. Preferably, according to the invention, the label comprises several identical or different target polynucleotides, preferably different target polynucleotides. The target polynucleotide may be referenced to a confidential target/product database that establishes an association between the target polynucleotide and the tagged product.

By "confidential database" is meant a database that is accessible to only the maker of the invention and/or only the maker/creator of the product for a given product (hereinafter the maker and/or the maker/creator of the product will be referred to as the "implementer of the invention"). This is the corresponding basis for assigning a specific identification feature (signature) determined according to the invention to each product or product family.

In the case where only the target polynucleotide and the decoy polynucleotide are used as the identifying characteristics of the product, the data or corresponding basis may be referred to as a "target/product secret basis" or a "target/product basis". Thus, if we wish to authenticate the product of the invention, one must first search for the target sequence assigned to the product in this target/product base and then find out whether the target sequence is actually present in the product, for example by searching using one of the methods described below. If the target sequence is actually present, the product is judged to be authentic. However, if the target sequence is not actually present, it can be concluded that the product is a counterfeit. Of course, this is only valid if all genuine articles are marked according to the invention. The correspondence basis may be constructed by the manufacturer of the identification features of the invention and/or by the manufacturer/creator based on the list of identification features of the invention, for example by having each identification feature corresponding to a certain product. In this case, the authentication is direct.

In the present invention, a "decoy polynucleotide" refers to a polynucleotide whose sequence has been selected and constructed to be different from a target sequence. The decoy sequence is used to be intermixed with the indicia of the present invention but is not used to be searched in a product in order to authenticate the product. Decoy sequences are used to complicate the work of counterfeiters attempting to replicate the identifying features of the present invention. In fact, the target sequences are known only to the practitioner of the invention. According to the invention, if the decoy, target and decoy sequences are short sequences that are single stranded and do not hybridize to each other, it becomes more difficult (if not impossible) to distinguish between one or more specific sequences in a mixture of target and decoy sequences for replication (in this case, to distinguish between target sequences for replication of the target). The more decoys, the more difficult it is to replicate the identifying features in the entity, and statistically, the lower the chance of randomly determining the sequence of the target polynucleotide. The sequences of these decoy polynucleotides are also known to the practitioner of the invention, however, these polynucleotides are not in the database to be assigned to the product.

In the present invention, the "recognition polynucleotide" refers to a polynucleotide having a predetermined sequence and length that enables identification of a target polynucleotide. The identification polynucleotide, which has properties or is present at a concentration that facilitates rapid retrieval and identification, serves as a first authentication test: the absence of which is the first sign of a counterfeit. An identification polynucleotide is one or several polynucleotides whose sequences have been selected and constructed to constitute a code for use in a confidential database or "target identification basis" for identifying target polynucleotides, which "target identification basis" is used to give information about the number, nature, sequence and length of target polynucleotides. When several identification polynucleotides are present, they may be referred to as "identification polynucleotide groups". In this case, the target polynucleotide or group of target polynucleotides determined according to the invention is assigned in the basis of the target identification of the respective identification polynucleotide or group of identification polynucleotides. The sequence of the recognition polynucleotide is known only to the practitioner of the invention. The presence of the identification polynucleotide in the identification feature of the invention is optional and corresponds to a particular embodiment of the method of the invention. If one or several identification polynucleotides are used in the identification feature of the invention of the product, a confidential basis for identifying the target is created by the practitioner of the invention. This basis makes it possible to identify the target polynucleotide present in the product to be authenticated on the basis of the identification polynucleotide. In this case, the authentication of the product is indirect. In fact, if it is desired to confirm that evidence assumes a product having the identifying characteristics of the invention, the identifying polynucleotide is identified according to one of the methods of the invention described below, followed by searching the target identification database for the target sequence assigned to the product, and then searching whether the target sequence is actually present in the product, for example by using one of the methods described below. If the target sequence does exist, the product is judged to be authentic. However, if the target sequence is not present, the product can be determined to be a counterfeit. It should be noted that in this embodiment of the invention, there are two steps of identifying the polynucleotide prior to authenticating the product, which complicates the task of a potential counterfeiter. This is of course only valid if the genuine article is marked according to the invention. According to the present invention, the sequence of the recognition polynucleotide may contain subsequences carrying codes that enable the user of the present invention to discover which are target polynucleotides and which are decoy polynucleotides based on the basis of target recognition. Thus, in the authentication process of a product, these identification polynucleotides are extracted from the product to be authenticated or directly identified in or on said product. Once the nature of the identification polynucleotide is detected (for example, but not limited to, identification of the target polynucleotide may be achieved by reading the sequence of the identification polynucleotide, or by identifying an array of identification polynucleotides present in a plurality of putative encoding polynucleotides), the user of the present invention may refer to the corresponding table (target polynucleotide identification basis) and read therein the nature of the target polynucleotide theoretically present in the product to be authenticated. The table may be stored, for example but not limited to, in a secure computer database (enabling confidentiality to be ensured), which is created during the marking of the product, and in which the pairs appear: "code for read identification polynucleotide" - "target polynucleotide for search". Thus, upon referring back to the table, a user of the present invention can infer the exact nature of the target marker that should be present in the product to be authenticated. Thus, the target polynucleotide is extracted and then identified. If the detected target polynucleotide exactly matches the theoretical code read in the table, the product may be authentic. The user of the present invention may be suspected of labeling a mixture of products if other target polynucleotides are present. If the detected target polynucleotide is completely different from the desired polynucleotide, it may be a counterfeit. If no recognition polynucleotides are used in the recognition feature of the present invention, only the target/product database is useful.

In the present invention, the polynucleotides of the plurality of polynucleotides may be designed, for example, by methods known to those skilled in the art, for example, using software that performs, for example, the algorithms set forth below, such that for a given polynucleotide selected from the plurality of polynucleotides that make up the marker, none of the other polynucleotides of the plurality or the reverse complement of any of these polynucleotides is comprised of the sequence of the polynucleotide that is complementary to the given polynucleotide. Thus, the polynucleotides of the recognition features of the invention will not form a duplex complex, e.g., a nucleic acid duplex (such as a DNA duplex), nor any hybridization between them, including in the temperature and molecular regulatory environment of the product, and in the temperature and development environment of the polynucleotide molecule.

"formation of a duplex complex" refers to the pairing of complementary nucleotides that includes thermodynamic stability under the conditions described above.

"reverse complement polynucleotide" of a given polynucleotide refers to a novel polynucleotide, either present or theoretical, in which each nucleotide of the given polynucleotide is replaced by a complementary nucleotide capable of pairing with that nucleotide, e.g., in the case of a deoxyribonucleic acid polynucleotide, adenine for thymine, thymine for adenine, cytosine for guanine, or guanine for cytosine.

"hybridization" refers to the binding of two complementary single-stranded polynucleotides by non-covalent bonds. The hybridization may be complete, i.e., the sequences are fully complementary, or incomplete, i.e., the sequences are not fully complementary, but are complementary enough to hybridize to each other and form a double-stranded structure.

In the present invention, "non-hybridization" means that two single-stranded polynucleotides are not bound by a non-covalent bond because the two single-stranded polynucleotides are not complementary and/or are not complementary enough to form a double strand.

It is noteworthy that not all of the various polynucleotides of the markers of the invention to be introduced into a product or substance of interest are assigned to a database for direct or indirect authentication of the product. Thus, it is possible to use only a limited number of target polynucleotides which are the polynucleotides to be sought in the product authentication process and to introduce them into the product simultaneously with a large number of decoys and, if necessary, identification polynucleotides whose sequences do not correspond to those of the target polynucleotides. Thus, the target polynucleotide can be "swamped" into a large number of decoy polynucleotides, which can be particularly confusing in the case of malicious attempts to decode the target polynucleotide with the purpose of replicating the identifying feature. In addition, according to the particular embodiment of the invention in which an identification polynucleotide is used, merely reading and decrypting the code carried by the identification polynucleotide can ascertain which in the combination of target and decoy polynucleotides actually correspond to the target sequence, and by excluding ascertaining which are merely decoys intended to mislead counterfeiters.

The labeling method of the present invention can be carried out using a label which is deoxyribonucleic acid and/or ribonucleic acid. Thus the polynucleotides of the plurality of polynucleotides may be single-stranded deoxyribonucleic acid sequences or single-stranded ribonucleic acid sequences, or a combination of deoxyribonucleic acid sequences and ribonucleic acid sequences. The marker of the invention may thus consist of the usual bases known as "natural bases", such as, for example, the bases present in DNA: adenine, guanine, thymine, cytosine; or the base present in the RNA: adenine, guanine, uracil, cytosine (see, for example, Molecular Cloning, Maniatis, Cold Spring-Harbor, second edition, pages C3-C14 [1 ]). The markers of the invention may also comprise natural or synthetic compounds called "modified bases" with a low frequency of occurrence, such as Dihydrouridine (DHU), inosine or pseudouracil, which are produced by modifications such as deamination of the aforementioned bases. Nitrogenous bases can be composed of natural isotopes and/or stable isotopes of different atomic mass and/or modified to establish a number of hydrogen bonds during the hybridization process that are different from normal hydrogen bonds.

According to an embodiment of the present invention, the sequence of deoxyribonucleic acid may include the four natural or modified bases A, C, G and T in the same ratio within its sequence. According to another embodiment of the invention, the sequence of ribonucleic acid may contain the same proportions of the four natural or modified bases A, C, G and U within its sequence. According to another particular embodiment of the invention (including the two preceding embodiments or not the two preceding embodiments), the set of polynucleotides comprising the label has the same number of nucleotides and the same molecular weight. Advantageously, this embodiment of the invention makes it more difficult, if not impossible, for a potential counterfeiter to isolate and authenticate the polymer. For example, it is not possible to isolate and characterize the identifying features of the latter embodiment, particularly the last embodiment, as follows: the separation and identification is carried out according to molecular size and/or molecular weight by means of techniques such as electrophoresis (for example on agarose gels or polyacrylamides) and/or mass spectrometry.

For example, in the labels of the present invention, a single-stranded polynucleotide (or oligomer) of 20 nucleotides (each nucleotide being selected from 4 possible bases) is capable of achieving 4²⁰Different sequences, i.e. about 1.1X 10¹²1 trillion combinations. Therefore, according to the present invention, the probability that a target marker is randomly extracted from a plurality of polynucleotides having a size of 20 nucleotides in the marker of the present invention and is a marker that has been assigned to a product in the target/product database is almost equal to zero. In addition, the marker of the invention consists of several target molecules of predetermined length and sequence, several decoys and, if necessary, several recognition polynucleotides, which ensure both a very high safety and a significant inviolability of the marker.

Thus, the polynucleotides used may comprise, for example, by their nature a directed combination of 4 nitrogenous bases, the nature of which is defined by the practitioner of the invention. The combinations that are the source of specificity of each polynucleotide of the markers of the invention and that can carry information about the labeled product can be calculated in a computerized manner according to the needs (coding complexity, type of information carried by the markers) and the physicochemical properties exhibited by these markers (hybridization, molecular weight, fragment size, composition of nitrogenous bases).

Depending on the product to be labelled and the technique of detection of the label in question, one or several target polynucleotides may be used. Thus, the present invention allows for a considerable number of variations/alternatives of the identification features or indicia. By way of non-limiting example, the following different forms of the first group of target nucleotides can be cited:

-one or several single-stranded target polynucleotides of large size, i.e. comprising for example about 500 to 5000 nucleotides or bases;

-one or several single-stranded target polynucleotides of small size, i.e. comprising for example about 5 to 200 nucleotides or bases, such as 5 to 50 nucleotides or bases;

-one or several single-stranded target polynucleotides of average size, i.e. comprising for example about 201 to 499 nucleotides or bases;

one or several single-stranded target polynucleotides having a constant end sequence and a variable end sequence,

one or several single-stranded target polynucleotides inserted (or defined) in a large-sized polynucleotide, such that these inserted target polynucleotides form a larger part of the polynucleotide,

one or more single-stranded cyclic polynucleotides, or

-a combination of these different forms.

According to the present invention, at least two target polynucleotides may be used, one being a circular polynucleotide and the other being a linear polynucleotide. Depending on the complexity of the labeling chosen by the practitioner of the invention, a plurality of target cyclic or linear polynucleotides, or mixtures thereof, may be used.

According to a particular embodiment of the invention, when some or a group of single stranded polynucleotides are linear, they may comprise variable ends which are variable between different polynucleotides and constant ends which are constant between different polynucleotides. "constant end" refers to a portion of a polynucleotide sequence that includes one of the two ends of the polynucleotide sequence and exhibits a predetermined and constant sequence, that is to say is the same for part or all of the target sequences of the marker of the invention. "variable end" refers to the portion of a polynucleotide sequence that includes the other of the two ends of the polynucleotide sequence and exhibits a predetermined sequence that is variable between different target sequences in a marker of the invention. This provides a particular advantage of the target polynucleotide. In fact, as described below, for the purpose of detecting and decrypting the label for the purpose of authenticating genuine products, a solid phase substrate for decryption on which a polynucleotide complementary to the variable end of the target polynucleotide is immobilized can be used for the micro DNA array. The constant ends themselves can be used to highlight hybridization of a target polynucleotide on a solid phase substrate, for example, with the aid of biotin/streptavidin. The detection mode is described below.

The number and nature of the target polynucleotides and their size make it possible to define the complexity of the labeling and, in combination, the number of possible combinations. The number of possible combinations increases exponentially with the size of these polynucleotides. By selecting an identifying feature consisting of one or several polynucleotides of all polynucleotides of the same size, a large number of combinations can be envisaged. The probability of randomly replicating an identifying feature in all possible combinations is almost zero.

According to the invention, the marking information may consist in:

-target polynucleotide sequences, each target polynucleotide sequence being assigned to a product in a target/product database;

and/or

-in one or several combinations of target polynucleotide sequences, said combinations are assigned to products in a target/product database.

Thus, several target polynucleotides having predetermined sequences can be used. A given set of products may also be tagged by, for example, selecting a population or "library" of 20 polynucleotides all having different predetermined sequences, and selecting a combination of, for example, 10 of these 20 sequences for each product.

Decoy polynucleotide sequences are added to these target sequences to constitute the markers of the invention. According to the invention, decoy polynucleotides do not hybridize to the target polynucleotide, and they function to make decryption of the marker of the invention for replication more difficult for counterfeiters. As noted above for the target polynucleotide, these decoy polynucleotides may be in linear or circular form, or a mixture of circular and linear polynucleotides. The number of decoy polynucleotides added to the tag depends on the desired interference. Preferably, the number is higher than the number of target polynucleotides. Examples are given above. The decoy polynucleotides are of the same or different length to each other, preferably of the same length as the target sequence present in the marker of the invention, e.g. as indicated above for the target polynucleotide, of 15 to 5000 bases, e.g. 15 to 200 bases, e.g. 20 to 200 bases, e.g. 201 to 499 bases, e.g. 500 to 5000 bases, or a mixture of these lengths.

Recognition polynucleotides can be added to these target and decoy sequences, enabling discrimination between target and decoy polynucleotides by means of a database as described above. The number of polynucleotides identified will depend on the complexity of the desired label. The recognition polynucleotide may be circular or linear. They may be of the same or different length to each other or of the same or different length to that of the target and decoy polynucleotides, as noted above for the target polynucleotide, and may be 5 to 5000 bases in length, for example 15 to 200 bases, for example 20 to 200 bases, for example 201 to 499 bases, for example 500 to 5000 bases, or a mixture of these lengths.

Thus, in the tags of the invention, the polynucleotides in the plurality of polynucleotides may be cyclic, linear or a mixture of cyclic and linear polynucleotides, e.g. having a free 3 'OH end and a free phosphate 5' end. Preferably, the labelled polynucleotides of the invention are 5 to 5000 nucleotides, such as 5 to 100 nucleotides, such as 5 to 50 nucleotides, such as 20 to 50 nucleotides in length.

According to the invention, said polynucleotide of the plurality of polynucleotides is a single-stranded polynucleotide. In fact, one of the features of the present invention is the use of a single chain that makes decrypting the mark of the present invention more difficult.

Thus, the marking method of the present invention enables the production of extremely large quantities of markers. Each marker comprises a code consisting of a target polynucleotide and, if necessary, an identification polynucleotide.

The sequence of the polynucleotide of the marker of the invention may be created empirically or, preferably, in particular for the sake of rapidity, by means of suitable software which can be generated for this purpose. In the latter case, it is the computer Design or "electronic Design" (Design In silica) of the marker of the invention.

In order to determine the sequence of the target and decoy polynucleotides and, if necessary, the recognition polynucleotides of the markers of the invention, the following algorithm can be used:

- (0) creating a set E containing the set of polynucleotides resulting from the label.

- (1) randomly generating a first polynucleotide p, wherein the size and number of polynucleotides of said first polynucleotide can be defined by the user, p not belonging to E.

- (2) calculating the hybridization score between the following polynucleotides according to the algorithm of Smith and Waterman [2 ]: polynucleotides resulting from the linkage (ligation) of two polynucleotides p on the one hand and of two polynucleotides selected from the group E of polynucleotides and their reverse complements on the other hand.

- (3) if a set of scores does not exceed the threshold given by the user, adding p in E. Returning to step (1) provided that E is not the desired size. These thresholds are the minimum alignment score above which two sequences are considered to have sufficient identity to hybridize to each other.

Once the sequences of the target and decoy polynucleotides are determined, they can be made by any existing method known to the skilled person. Depending on the nature of the polynucleotide being produced and on the marker chosen, one or several strategies may be used: a cyclic and/or linear single-stranded ribonucleic acid and/or deoxyribonucleic acid having a variable size of, for example, 5 to 5000 bases is synthesized. By way of examples of available strategies for carrying out the present invention, a strategy [3] capable of synthesizing circular single-stranded polynucleotides or an electronic synthesis strategy [4] of polynucleotides may be cited.

Where identification of polynucleotide sequences is required for tagging, their respective sequences may be determined empirically or by algorithms as previously described such that the identification polynucleotides do not hybridize to each other, nor to the target or decoy polynucleotides.

Labeling of solutions or compounds according to the methods of the invention with ribonucleic acid or deoxyribonucleic acid labels can be accomplished in a variety of ways depending on the complexity of the label desired. Each target polynucleotide carries specific information inherent to its sequence. Each target polynucleotide or combination of target polynucleotides can be used in a unique manner.

The first possible encoding level may be the level at which one or more target polynucleotides are used. Several batches of tagged products can thus be traced by one or several polynucleotides of defined size and predetermined sequence, but different from each other.

A second possible level of encoding is to use several target polynucleotides selected in an initial pool of target polynucleotides. Thus, the code is no longer from the individual sequences of the target polynucleotide, but from the combination of target polynucleotides found in the product. Thus, according to the invention, the tagged product may be tagged with N target polynucleotides, said N target polynucleotides being selected from a possible number N of different polynucleotides, N being comprised in the interval [ 0; n ] in the formula (I).

In all cases, the labeling of the invention can be accomplished by essentially using a third level of encoding of the recognition polynucleotide, whereby, if the product is authentic and labeled using the method of the invention, it can be indicated with the aid of a target recognition database which labels are present in the target polynucleotide which are readily available to the manufacturer and which may have different properties and concentrations.

According to the invention, the steps of the method for marking the product may be objects having a specific tracing capacity. The tracing ability can be ensured by introducing specific information for each step in one or several confidential databases.

Thus, according to the invention, each batch of markers or combination of markers can be identified, for example, by alphanumeric identifiers. The identifier may appear on the product or through any optical representation mode: such as, for example, a bar code, a data matrix, etc., on the container of the marker batch. The identifier may also serve as an index into the first database in which information of the marker lot is stored, such as: the sequence of each marker making up the marker batch, the respective ratio of the number of each marker, the date of manufacture, etc.

Each container of a marker batch tracked by means of an identifier of the marker batch may be associated in a database with reference information, e.g. a customer order, or with delivery reference information for delivering the container to the customer. Confirmation of receipt from the customer may also be entered into the database.

The nucleic acid does not change any physicochemical properties of the marked product. In addition, the nucleic acid does not have any influence on its container, i.e. the marked product. Finally, nucleic acids have proven to be very stable in many experiments performed by the present inventors. This stability is demonstrated in the examples below.

By way of non-exhaustive, the single-stranded DNA and RNA markers encompassed by the present invention may be included in a wide range of products and substances that are susceptible to being targeted for illegal, abusive copying (counterfeiting), illegal transactions on the black market, and/or in products and substances for which tracking of a trajectory is critical (product tracking).

The method of the invention is applicable to the marking of all liquid, semi-liquid or solid industrial or consumer products. The following products may be cited, but are not limited to, for example: fragrances, cosmetics, sanitary products, foodstuffs, condiments, plant extracts, tobacco products, beverages, textiles, leather products, pharmaceuticals, powders, varnishes, inks, coatings, chemical products and compounds, more generally, all goods and products which are susceptible to counterfeiting.

The invention can also be applied to the whole range of products from the high-end industry and the cosmetics industry: perfumes, eaux de parfum, colognes, essential oils, creams, masks, pomades, etc.

In the field of industrial and consumer products, the invention can also be used for tracking various substances such as inks, resins, varnishes, paints, dyes, additives, fragrances (aroma), glues, powders, etc.

In the field of food industry, the invention can be applied to high-end products that may be the target of counterfeiting or fraud (mixing), such as in particular alcoholic beverages, spirits, super wines, or even any product for which it is important to ensure authenticity, for example for safety reasons.

Markers may also be used in the medical industry to mark and track drugs and other medications.

The present invention may also be used to track biological samples in a hospital environment. For example, implementing traceability strategies for blood samples in biochemical laboratories, tumor samples in patho-anatomical laboratories, or strategies aimed at building directly certified human tissue banks that can be maintained and tracked in biological resource centers for many years.

In general, a product may be marked as the item it represents, or as a component. For example, paper documents may be marked with ink that has been used on the paper document and that has been previously marked.

According to the invention, the step of adding the marker of the invention may be achieved by any suitable means, thereby enabling the addition of the polynucleotide constituting the marker of the invention to the product to be marked. The products and substances to be marked can be marked in bulk by introducing the markers studied and provided at their final concentration, or on the surface of the products. The label is a polymer of ribonucleic or deoxyribonucleic acids with physicochemical properties derived from the properties of ribonucleic or deoxyribonucleic acids: they are negatively charged hydrophilic molecules. Depending on the nature of the products to be marked, they may be pre-diluted or added directly to the product. They may also be encapsulated. Even if they are deposited or integrated on the surface of the product.

The addition of the marker of the invention to a product may be achieved by adding the plurality of polynucleotides to the product or to or on the final product (i.e. on the surface) during manufacture of the product. The invention therefore also relates to the products obtainable by the marking method of the invention.

According to the invention, when the addition of the marker is effected at the surface of the product to be marked, it can be carried out, for example, by dipping the final product into a solution containing said marker or by spraying or evaporating said solution on the final product. Preferably, the solution is a protic solvent such as ethanol, methanol or diethylene glycol, or a polar aprotic solvent such as acetone or tetrahydrofuran. This mode of addition is suitable, for example, for post-manufacture solid products such as fabrics, leather, wood, paper, cardboard, tobacco products, cigarettes, cigars, etc.

The addition may also be made during the manufacture of the product by mixing the marker with the compounds or ingredients that make up the product. The introduction of the plurality of polynucleotides may be effected on or in the ingredients of the product. This type of addition is suitable for any product and substance that undergoes a liquid or semi-liquid phase in manufacture, to which the marker may be added. It may be the case, for example, that the cosmetic or pharmaceutical product is integrally marked.

According to a specific embodiment of the present invention, the step of encapsulating the plurality of polynucleotides in a lipid carrier may be performed before the step of adding. This encapsulation step enables the polynucleotide to be maintained in a favorable environment, or facilitates its future extraction. For example, as the encapsulation product, a product selected from the group comprising cationic lipid carriers, such as dioleoyloxypropyltrimethylammonium bromide (DOTMA) and Dioleoylphosphatidylethanolamine (DOPE), or polynucleotide complexes having molecules such as polylysine, protamine or Polyethyleneimine (PEI), which are called polymeric complexes (polypex), may be used. For example, bioch. biophys. acta 1280: [5], j.biol.chem.269: 2550[6] or AAPS PharmSci.2001; 3(3): e21[7 ].

According to another embodiment of the present invention, the step of encapsulating and/or protecting the plurality of polynucleotides in a lipid carrier or other carrier may be performed before the step of adding. This step further ensures stability and/or facilitates its recovery.

In the present invention, "protection" means protection of the polynucleotides of the invention, in particular from any physicochemical attack from the environment, wherein for example the identifying features of the invention (flavour, food) are found filled in carbon nanotubes in these polymers.

Whatever mode of addition is chosen, it is preferred that the marker of the invention be added to the product to mark it at very low concentrations, from micromolar to femtomolar. These labels are added at variable final concentrations, depending on the existing or upcoming detection technique under consideration and the size of the labels, and the respective concentrations of each target polynucleotide of the first set may differ and constitute a coding subset. According to the present invention, the concentration of the plurality of polynucleotides after addition to the product may be, but is not limited to, 10^-6Mol/dm³～10^-18Mol/dm³. For liquid or solid volumes, it is suggested to use such amounts/volumes. For liquids, it corresponds to the amount of marker mixed with the product in volume units. For solids, it corresponds to the amount of marker mixed with the product or deposited at the surface of the product in volume units. When referring to the surface of the product, also by unit/area, i.e. 10^-6Mol/dm²～10^-1Mol/dm²To define said amount. These concentrations can be achieved by diluting more concentrated solutions. The solution may be as defined above.

The invention also relates to a method for detecting a product marker obtainable by the marking method of the invention, said method comprising an analysis step of a plurality of polynucleotides, which analysis step enables the specific detection of at least one target polynucleotide.

In addition, the present invention provides several methods for detecting the markers of the present invention. The detection can be carried out outside or inside a laboratory, for example by means of a portable system (for example by means of a DNA microarray designed in particular for detecting the target polynucleotides of the markers of the invention).

According to the invention, the analysis step can be carried out, for example, by immunoassay. According to the invention, the analysis step may comprise, for example, a step of sequencing the encoding polynucleotide. According to the invention, in particular when the target polynucleotide is a ribonucleic acid, the analyzing step may for example comprise reverse transcription of the ribonucleic acid into deoxyribonucleic acid. According to the invention, the analysis step may comprise a colorimetric, luminescent or fluorescent detection in combination with specific hybridization. In other words, the analysis step may use a particular means of detecting the target polynucleotide.

The analytical techniques used in accordance with the present invention can exploit the physicochemical properties, coding and specificity of the labels. The assay techniques may be associated with sandwich assay techniques, detection techniques using DNA microarrays, or any other technique known to those skilled in the art that can detect the presence of and/or identify polynucleotides.

In a particular embodiment of the invention, the target polynucleotide and decoy polynucleotide may be small-sized polynucleotides, i.e. 8-30 nucleotides in length per nucleoside, and single-stranded. According to this embodiment and advantageously, depending on the nature of these polynucleotides, they cannot be used as template strands that can be amplified and detected by exponential amplification techniques such as the polymerization chain reaction ("PCR", polymerase chain reaction). Therefore, it cannot be detected by PCR amplification. In this embodiment, detection of the polynucleotide can be performed by hybridization methods and direct detection without amplification and can only be accomplished by a user who knows the identification characteristics used. In addition, counterfeiters who attempt to extract, amplify and replicate the various polynucleotides present in the tagged product are discouraged. In addition, it is advantageous that a detection method comprising only a hybridization step and a detection step of said hybridization can be carried out much more quickly. The polymerase chain reaction takes several hours, whereas simple hybridization of the polynucleotide is almost instantaneous as long as the detection is specific for the polynucleotide to be detected.

According to the invention, the analytical technique used may be based on the physicochemical properties, coding and specificity of the label. It may comprise exponential or linear amplification of the target polynucleotide, and any other technique may be used to detect the presence of the label. In other words, the analyzing step may comprise a linear amplification step of the target polynucleotide.

Prior to the analyzing step, the method of the present invention may further comprise the steps of:

(a) sampling the product; and

(b) extracting a plurality of polynucleotides of the product.

Here, the method consists of a method which enables the detection of the marking of the invention by sampling the product.

After extraction, the presence of the marker can be analyzed as described above.

According to the present invention, the extraction step (b) may be achieved by any technique known to the skilled person, thereby extracting polynucleotides from the sample. The polynucleotide can be extracted according to a strategy that depends on the nature of the tagged product. Any type of ribonucleic acid or deoxyribonucleic acid extraction technique, known or that may be forthcoming, may be used to extract polynucleotides from a plurality of tagged products. It may be, for example, a phenol-chloroform extraction. Examples of extraction techniques that can be used in the present invention are described in Molecular Cloning, Maniatis, Cold Spring-harbor, second edition, pages E3-E4 [8 ].

The method of analysis of the marked product may thus consist of the following steps: extracting polynucleotides from these products, detecting the code carried by the identified polynucleotides, accessing a database to identify the target polynucleotides, thereby decrypting the code carried by the target polynucleotides, then using this information to find the target polynucleotides among a plurality of target polynucleotides including decoy polynucleotides, then detecting the presence of the target polynucleotides, which is characteristic of the tagged product (in the case where a tag is detected) or concluding that the product is counterfeit (in the case where the target polynucleotides are not present or the tag is not consistent with the target/product database).

According to a particular embodiment of the invention, the detection specific to the analysis step may comprise, for example, the following successive steps:

(1) contacting a plurality of polynucleotides with a solid phase substrate having probe sequences immobilized thereon, which probe sequences are complementary to one of said ends of said at least one target polynucleotide of said labeled plurality of polynucleotides of said product, said contacting enabling immobilization of the target polynucleotide on said carrier by hybridization to a complementary probe sequence immobilized on said substrate;

(2) (ii) removing polynucleotides not hybridised by step (i); and

(3) detecting the presence of the target polynucleotide on the substrate.

In the present invention, the detection performed in step (iii) may be achieved by modified specific means, which may for example involve detection using fluorescent molecules, detection using luminescent molecules, detection using enzymes whose reaction products may be coloured, detection using enzymes whose catalysed reaction is an exothermic reaction, detection using enzymes whose catalysed reaction emits light, or detection using proteins (e.g. antibodies, enzymes) specific for the target polynucleotide.

According to a particular embodiment, the specific detection also comprises, between steps (i) and (ii), a step of capturing said plurality of polynucleotides on a substrate by means of at least one specific target polynucleotide capture system on which at least one target polynucleotide of the labeled plurality of polynucleotides of said product is immobilized.

When the label of the invention comprises an identification polynucleotide, the method of this embodiment may further comprise, before step (i), a step (x) of identifying these identification polynucleotides and a step (y) of selecting a solid phase substrate on the basis of the identified identification polynucleotides, the solid phase substrate thus selected comprising a probe sequence complementary to a target sequence identified on the basis of the identification polynucleotide.

Advantageously, the detection method can be used with a marker of the invention comprising a target polynucleotide having a constant end and a variable end. These target polynucleotides are as defined above. According to the invention, polynucleotides complementary to the target polynucleotide (referred to as probe sequences) can be immobilized on a solid substrate by any means known to the skilled person. These on-substrate detection techniques and the types of substrates that can be used in the present invention are described in Molecular Cloning, Maniatis, Cold Spring-Harbor, second edition, pages 9.47-9.57 [9 ]. For example, the immobilization of the probe sequence on a matrix may be performed by means of a biotin/streptavidin linkage, the probe being coupled to a biotin molecule, and the matrix displaying streptavidin molecules. Immobilization of the probe sequence can also be achieved, for example, by forming covalent bonds with a charged nylon membrane, which forms a solid phase matrix. These techniques that can be used in the present invention are, for example, those described in publications [10], [11] and [12 ]. In other words, a polynucleotide complementary to a target polynucleotide is an example of a capture system specific for the target polynucleotide sought.

According to the present invention, a target polynucleotide immobilized on a substrate by hybridization with a probe sequence can be detected by any suitable means known to the skilled person. It may be, for example, detection using a polynucleotide labeled with a labeling agent capable of being selected from the group consisting of a fluorescent dye, a colloidal gold particle and an enzyme and complementary to the other end of the target polynucleotide.

This mode of detection on a solid phase matrix allows for easy, reproducible and immediate detection of the markers of the invention. Which can be advantageously used in the present invention.

The detection method of the invention may further comprise a step of comparing the results of the step of analysing the target polynucleotide with the contents of a database enabling identification of the target polynucleotide, and a target polynucleotide analysis enabling identification and authentication of the product and also enabling determination of the origin of the product. In other words, the database and decryption of the code carried by the encoding polynucleotide enables identification of counterfeits from the original product.

By finding the target polynucleotide contained in the tagged product using the analytical technique used to use the target polynucleotide contained in the tagged product, information about the product can be found. The absence of a target polynucleotide that should be present in the product being tested indicates a possible counterfeit of the product. The detection of several different markers or recognition polynucleotides of the invention in the same product may indicate that this is a result of an abnormal initial mix (abnormal initial mix).

During the manufacturing step of the product to be marked, the manufacturer user of the marker of the invention can be associated with an identifier of the container of the marker batch, reference information relating to, for example, the product batch marked with the marker batch. The association may be made, for example, in the same or a different database than the aforementioned database. Depending on the information system used by the user, it is preferred that said information entered into the database and related to the production lot is sufficient to enable the lot to be tracked unambiguously.

When a product sample suspected of being counterfeit is analyzed, the detection of the identified polynucleotide sequence can be compared to information entered into the database during manufacture and delivery of the marker.

For example, if no marker is identified, it may be characterized as counterfeit. A mark can also be marked as counterfeit if at least one marker in a batch of markers that has been obtained by querying the database for the exact composition and has been partially revealed is missing. Counterfeiting can also be characterized if the tag that should be present in the batch tag has integrity, but the goods tested are not from the manufacturer that has received the order for the tag batch that was just disclosed.

In the case of an abusive distribution channel or parallel market, the product is genuine and the batch of the disclosed marker actually corresponds to the batch of the marker delivered to the manufacturer of the tested product. The method of the present invention is capable of obtaining an identifier of a tag batch from a manufacturing database tag upon request. This identifier advantageously enables the manufacturer to compare the theoretical allocation of the marked product lot to one of its customers with the actual allocation of what is suspected of being a parallel operation (parallelisms) noted during the sampling of the product, as long as the tracer system allows it. If the theoretical distribution of the product does not match the actual distribution, there may be abuse of the distribution channel.

Other advantages will become more apparent to those skilled in the art upon reading the following embodiments, which are illustrated by the accompanying drawings given by way of illustration.

Drawings

FIG. 1 shows the cleavage site of NbBpu 10I.

FIG. 2 shows the addressing of probes on a microarray.

Fig. 3 shows an emission spectrum and an absorption spectrum of Cy 5.

FIG. 4 shows a method for detecting a marker of the invention on a substrate: markers M1 and M2 are present in a mixture. They create a "bridge" between the probes immobilized on the microarray and the universal probes: thus, a signal (fluorescence of Cy5) was detected.

Fig. 5 shows the detection of the marker by a NanoChip workstation (registered trademark) (Nanogen Inc.).

FIG. 6 shows the principle of coupling labels.

Fig. 8 shows a diagram of detecting the marking of the present invention and authenticating the marked product of the present invention.

Detailed Description

Examples

Example 1: manufacture of a marking and marking of a product according to the method of the invention

In this example, the target polynucleotide and decoy polynucleotide used are single-stranded deoxyribonucleic acid sequences of 28 nucleotides in size. The encoding polynucleotide is a circular DNA sequence of 4.3 kilobases (kb) in size.

The marked product is an essence solution: j' Adore perfume (registered trade mark, Christian Dior perfume).

3 flags are done, illustrating that there are 3 possible assumptions in the authentication method: an instance in which the product is authenticated and two instances in which the product is not authenticated.

Design of A target markers and decoy polynucleotides

Principle of 1.a.1

10 polynucleotides of a single-stranded deoxyribonucleic acid are produced as follows: the 5' nucleotides of the 10 polynucleotides are all identical, as defined by the user. It is generally the sequence GCAACTCCAG. The algorithm set out in the summary of the invention was then used to generate 18 3' nucleotides by using the following parameters: a string (word) equal to 18 nucleotides in length, 5G nucleotides, 5C nucleotides, 4 a nucleotides and 4T nucleotides.

Each novel polynucleotide is then randomly generated such that it contains the predetermined number of each base. The novel polynucleotide and the validated set of polynucleotides and the alignment score between the polynucleotides resulting from pairwise linkage of each polynucleotide in the set were calculated according to the Smith and Waterman algorithms using the following parameters (this step is not necessary for the first polynucleotide):

and (3) AT scoring: 1 matching

And (3) GC scoring: 1.5 matching

Mismatch penalty: -3

Gap penalty: -2

The minimum selection score is 3, meaning that the novel polynucleotide is aligned with a polynucleotide from the set, or a linker from two polynucleotides in the set, and excluded if the score is greater than 3. Otherwise, it is validated and added to the set of polynucleotides. This procedure was repeated until 10 polynucleotides were obtained.

Table 1.1 shows a list of these markers.

Table 1.1: sequence of the marker

The first 5 polynucleotides (primer-1 to primer-5) were considered as target polynucleotides. The last 5 (primer-6 to primer-10) were considered as decoy polynucleotides. These markers were then injected into the perfume along with the coded marker to a final concentration of 10^-12Mol/dm³。

Materials of 1.a.2

Synthetic Polynucleotide (Eurofins MWG GmbH), 100 picomoles/. mu.L

-H₂O, deionization, nuclease-free

A.3 Process

The 10 polynucleotides were mixed in three pre-labeling solutions and diluted in nuclease-free deionized water to a final concentration of 10 per label^-9Molar marker/liter (i.e., total marker is 5X 10)^-9mole/L). For each solution, each marker was pre-diluted twice with 1/100 in nuclease-free water (i.e., 1 μ L pipetted using a P10 pipette and 99 μ L pipetted using a P100 pipette), an intermediate dilution of 1/10000. Then, 10. mu.L of each of the intermediate dilutions was added to a 1.5mL test tube (Eppendorf, registered trademark), and then 50. mu.L of water (using a P100 pipette) was added to obtain 100. mu.L of a mixture. Table 1.2 shows three pre-labeling solutions (A, B, C) each containing a combination of 5 target polynucleotides selected from 10 possible polynucleotides (target polynucleotides or decoy polynucleotides).

Table 1.2: combinations of markers

	A	B	C
				Primer-1	x		x
Primer-2	x		x
				Primer-3		x	x
Primer-4		x	x
				Primer-5		x	x
Primer-6		x
				Primer-7		x

Design of coding polynucleotides

Principle of 1.b.1

The encoding polynucleotide is a single stranded circular nucleic acid sequence equal to 4.3 kilobases (Kb) in length. The encoding polynucleotide is synthesized, for example, based on plasmid pBR 322. The polynucleotide contains a specific sequence of contiguous nucleotides (A, T, G, C) known to the user of the present invention. This constitutes a unique code that enables the user to later know which combinations of theoretical target markers are present in the tagged product.

In this example, the coding sequence is a 20 nucleotide portion, exactly upstream (5 ' side) of 50 bases of a known universal sequence which remains constant regardless of the coding sequence and whose sequence is 5'-CTGTAAGCGGATGCC-3' (SEQ ID NO: 11). The user has a correlation table that enables him to associate the coding sequence of the coding polynucleotide with the combination of target polynucleotides expected in the tagged product.

The plasmid was first digested with the restriction enzyme nb. bpu10i, which cleaved the single-stranded DNA molecule, thereby recognizing the recognition site represented in fig. 1: cleavage site of nb. The polynucleotide was then digested a second time with exonuclease III, releasing the nucleotide from the 3' hydroxyl end of the strand cleaved by nb.bpu10i (this nucleotide is circular), leaving the strand undigested by nb.bpu10i.

Materials of 1.a.2

Plasmid pBR322, Invitrogen (registered trademark)

-NbBpu 10I. (20U) No.: fermentas, # ER1681

-XR 10 buffer (buffer Nb. Bpu 101) No. # BR5(Fermentas)

Exonuclease III (1200U) number # ENO191(Fermentas)

-reaction buffer for exonuclease III; number # ENO191(Fermentas)

Ultrapure Phenol (trade Mark) buffer saturated Phenol, Invitrogen

-chloroform/pentanol (24: 1)

-3M sodium acetate

-95%, 75% frozen ethanol

-nuclease-free deionized water.

A.3 Process

●Digestion of

In a 1.5mL test tube (Eppendorf, registered trademark), the plasmid (5 μ g, i.e., 20 μ L) was added, 10-fold concentrated reaction buffer (40 μ L) was added using a pipette P100(Gilson Pipetman, registered trademark), water (399 μ L) was added using a pipette P1000(Gilson Pipetman, registered trademark), and the enzyme (1 μ L) was added using a pipette P10(Gilson Pipetman, registered trademark).

Table 1.3: digestion of pBR322

Plasmids	20μL
		X10 buffer R	40μL
Nb.Bpu10I(20U)	1μL
		H₂O	339μL

The tube was spun using a VTX-400 spinner (Labo model) and then incubated at 37 ℃ for 1 hour. DNA was extracted with phenol-chloroform according to the following strategy:

using a pipette P200(Gilson Pipetman, registered trademark), 1/2 volumes of phenol (200 μ L) and 1/2 volumes of chloroform (200 μ L) were added to the tube, followed by 10 seconds of rotation. Then centrifuged at 10,000rcf for 5 minutes (in a Centrifuge with a 1.5ml tube "Centrifuge 5415R" (Eppendorf, registered trade Mark)).

The aqueous phase was transferred to a new 1.5mL tube (Eppendorf, registered trade mark) and 1 volume (400 μ L) of chloroform was added. Spin and centrifuge at 10,000rcf for 5 minutes. This step was repeated 5 times.

Transfer the aqueous phase to a new 1.5mL tube, then add 1/10 volumes of 3M sodium acetate (40 μ L using P20) and 2.5 volumes of frozen ethanol (1000 μ L using P1000). Mixing and then incubation at-20 ℃ for 1 hour.

Centrifugation at 10,000rcf for 10 minutes. The supernatant was discarded and the pellet was washed carefully with 200. mu.L of 75% frozen ethanol. The precipitate is then dried in the open air. It was returned to 50 μ L (using pipette P100) of nuclease-free deionized water.

Treatment with exonuclease

In a 1.5mL tube (Eppendorf) containing 50 μ L of extract, the reagents of table 1.4 were added using pipettes P100, P20 and P200.

Table 1.4: linearization of pBR322

Reaction buffer for ExoIII	25μL
		Exonuclease III (1200U)	6μL
H₂O, nuclease-free	119μL

The tubes were mixed and then incubated at 30 ℃ for 10 minutes. The reaction was terminated by heating at 70 ℃ for 10 minutes.

-extracting the DNA as described in step 1.b.3 with phenol chloroform, precipitating the DNA as described in step 1.b.3, and then absorbing the polynucleotide into 20 μ L of nuclease-free demineralised water.

The labeling substance absorbed in 20. mu.L of water was dispensed using a Nanodrop spectrophotometer (registered trademark). The concentration was then returned to 10 by adding a sufficient volume of nuclease-free demineralised water using P10^-9mol/L。

I.C. labeling solution

Three flavour J' Adore solutions (registered trade mark, Christian Dior perfume) were so labelled. The polynucleotides are injected into a large number of products, the final concentration being 10 for the target and decoy polynucleotides^-12M, is 10 for an encoding polynucleotide^-12M。

The first solution is labeled with a pre-labeling solution a containing the target polynucleotides 1 and 2 and the decoy polynucleotides 8, 9, 10 and the encoding polynucleotides. The solution corresponds to the normal label in which the encoding polynucleotide is present, and/or the combination of the target polynucleotides actually corresponds to the information carried by the encoding polynucleotide.

The second solution is labeled with a pre-labeling solution B containing the target polynucleotides 3, 4, and 5 and decoy polynucleotides 6 and 7 and the encoding polynucleotides. This solution serves as an example of an incoherent label: despite the presence of the encoding polynucleotide, the combination of target polynucleotides is inconsistent with the information contained by the encoding polynucleotide.

The third solution is labeled with a pre-labeling solution C containing target polynucleotides 1, 2, 3, 4, and 5, no decoy polynucleotides, and no encoding polynucleotides. This solution also serves as an example of an incoherent label: on the one hand it does not comprise an encoding polynucleotide and on the other hand it contains other unexpected polynucleotides although it contains normally labeled target polynucleotides 1 and 2.

After labeling, the solution was stored at room temperature or 4 ℃.

I.D. extraction of polynucleotides

Before starting the identification of the coding and target polynucleotides, it is advisable to extract the marker from the alcoholic medium constituting the marked product (perfume).

1.d.1 principle

The marker is extracted from its alcoholic medium (perfume) and then recovered in an aqueous medium for the purpose of using molecular biological identification techniques. It is therefore preferred that the extraction must have a high yield (maximum amount of label recovered, ideally: 100% yield), but it must furthermore be preferred to free the label from all "contaminating" substances that might interfere with the detection technique.

Materials of 1, d.2

-phenol: ultrapure (trade Mark) buffer saturated phenol, Invitrogen

-chloroform/pentanol (24: 1)

-3M sodium acetate

-95% frozen ethanol (Carlo Erba Rectapur), 75%

-nuclease-free deionized water.

Method of 1.d.3

The technique used is a phenol-chloroform extraction technique. For this example, the marker was extracted from 500 μ L of labeled perfume.

To 500 μ L of labeled perfume in a 2mL test tube (Eppendorf, registered trademark), 1/2 volumes, i.e. 250 μ L, of phenol was added using a pipette P1000(Gilson Pipetman, registered trademark), then 1/2 volumes, i.e. 250 μ L, of chloroform was added using P1000(Gilson Pipetman, registered trademark), then 1/2 volumes, i.e. 250 μ L, of water was added using P1000(Gilson Pipetman, registered trademark), and then rotated for 10 seconds. Centrifugation was carried out at 10,000rcf for 5 minutes (in "Centrifuge 5415R", Centrifuge, Eppendorf, registered trade Mark).

The aqueous phase was transferred to a new 2mL tube (Eppendorf, registered trade mark) and 1 volume of chloroform was added. Spin and centrifuge at 10,000rcf for 5 minutes. This step was repeated 5 times.

Transfer the aqueous phase (above) to a new tube, then add 1/10 volumes of 3M sodium acetate (i.e. 50 μ Ι _ with pipette P100) and 2.5 volumes of frozen ethanol (i.e. 1250 μ Ι _, with P1000 in two portions). Mixing and then incubation at-20 ℃ for 1 hour.

Centrifugation at 10,000rcf for 10 minutes. The supernatant was discarded and the pellet was washed carefully with 200. mu.L of 75% frozen ethanol. The precipitate is then dried. The precipitate was placed in 20 μ L nuclease-free deionized water using pipette P20.

Detection of E encoding polynucleotides

Principle of 1.e.1

The information associated with the target polynucleotide carried by the encoding polynucleotide in the nucleic acid sequence of these encoding polynucleotides is first read (by sequencing techniques). This enables the user of the invention to know the exact nature of the target polynucleotide carrying the product authentication information by reference to the association table in 1. b.1.

Materials of 1, d.2

Universal sense primers (Eurofins MWG GmbH), 100 picomoles/. mu.L.

-5’-GGCATCCGCTTACAG-3’(SEQ ID NO：13)

Big Dye (registered trademark) terminator V3.1(Applied Biosystems)

-a template: solution of the extracted polynucleotide

Nuclease-free deionization H₂O。

1.e.3 Process

The sequencing reaction was carried out in a 200. mu.L tube (Eppendorf, registered trademark) according to the strategy summarized in Table 1.5, based on the solution of the extracted marker.

Table 15: sequencing reactions

Reagent

1 sequence

Big Dye (registered trademark)	2μL
		Polynucleotide (template)	5μL
Primer 10pM	1.6μL
		H₂O	q.s 10μL

The reaction was then carried out on the apparatus GeneAmp PCR System 9700(Applied Biosystem) simultaneously using the following cycles:

-96℃ 1’

-96℃ 10”|

-50 ℃ 5 "| > 25 cycles

-60℃ 4’ |

After column-based purification (Qiagen, registered trade Mark), the reactions were sequenced on a 16-capillary sequencer AB13100(Applied biosystems). Analysis of the results thus enables the user of the invention to read the nucleotide sequence of interest and to make an association between the read sequence and the target polynucleotide theoretically present in the tagged product.

I.F detection of target nucleotides

Principle of 1.f.1

A substrate (or DNA microarray) is used to detect the presence of a target polynucleotide. The matrix displays a series of several covalently immobilized probes, as well as the full reverse complement of the 3' variable region of the label. FIG. 2 shows the position of the probe on the chip.

When an extraction solution containing a putative label is brought into contact with the substrate, the label hybridizes to its reverse complement on the chip. After washing, the substrate is then contacted with a solution containing a probe (polynucleotide) coupled to fluorescein (Cy5), the sequence of which is the full reverse complement of the 5' universal region of the label.

FIG. 3 shows an emission spectrum (667nm) and an excitation (650nm) spectrum of Cy 5.

These probes will fix themselves to the substrate and will be detected later. Their presence allows the detection of the presence of the corresponding target polynucleotide in the starting solution. FIG. 4 shows the detection principle of the marker.

Material of 1.f.2

A reverse complementary probe of the marker-specific region (Eurofins MWG GmbH), 5' extended with a 20 base arm and biotinylated.

Probe anti-1 (SEQ ID NO: 14)

5’(B)-TGGATCCCGCACACGACTGACCATGACTCATGGAGTGC 3’

Probe anti-2 (SEQ ID NO: 15)

5′(B)-TGGATCCCGCACACGACTGATGACGTATGAGTCGCCAC 3′

Probe anti-3 (SEQ ID NO: 16)

5′(B)-TGGATCCCGCACACGACTGATGATAGAGTCCCCCTGAG 3′

Probe anti-4 (SEQ ID NO: 17)

5′(B)-TGGATCCCGCACACGACTGATGAGACTTGCCCTAGAGC 3′

Probe anti-5 (SEQ ID NO: 18)

5′(B)-TGGATCCCGCACACGACTGACTGAGATCCAGAGTCTGC 3′

Probe anti-6 (SEQ ID NO: 19)

5′(B)-TGGATCCCGCACACGACTGAGATGAGACCTCATGCTGC 3′

Probe anti-7 (SEQ ID NO: 20)

5′(B)-TGGATCCCGCACACGACTGAGAATGAGACTCCTGCTGC 3′

Probe anti-8 (SEQ ID NO: 21)

5′(B)-TGGATCCCGCACACGACTGAGAGTATTGCTGAGCCACC 3’

Probe anti-9 (SEQ ID NO: 22)

5′(B)-TGGATCCCGCACACGACTGATGAGATCACTGCCCTGAG 3′

Probe anti-10 (SEQ ID NO: 23)

5′(B)-TGGATCCCGCACACGACTGAGATAGAATGTGTGCCCCC 3′

-the polynucleotide reverse complement of the 5 'universal region of the label, coupled at 3' to fluorescein (Cy 5); (Eurofins MWG GmbH):

UnivFluo 5′CTGGAGTTGC-(CY5^*)3′(SEQ ID NO：24)

-solutions of extracted markers

Nanogen Automation Process (trade Mark) chip and cassette (Nanochip (registered trade Mark) Electronic Microarray, 100-Site Nanochip (registered trade Mark) Cartridge) blotted with the complement of the Probe variable region

-NanoChip workstation (registered trademark) molecular biology workstation (NanoChip Reader, NanoChip Loader)

L-histidine (Invitrogen, code: 0955061IX)

-multiple sorting filtration system (Millipore (registered trade Mark))

High salinity buffer (sodium phosphate 500mM, sodium chloride 500mM, Nanogen (registered trademark))

Low salinity buffer (sodium phosphate 50mM, Nanogen (registered trademark))

-NaOH 0.1M

1, f.3 method

Preparation of the Cartridge

The probe complementary to the specific region of the label was purified by a multi-sort filtration system (by Millipore (registered trademark)), and then absorbed into 60. mu.L of 50mM buffer L-histidine. And then transferred to a cassette. Each of them is addressed to a specific location of the box (by the workstation management policy) during 120 seconds. The biotin in 5' is immobilized on streptavidin of the substrate.

Table 1.6: blot of probes

	1	2	3	4	5
						1	Anti 1	Anti 2	Resistance 3	Anti 4	Anti-5
2	Anti-6	Anti 7	Resist against8	Anti-9	Anti-10

Hybridization of the labels

The cassette was rinsed twice with high salinity buffer (75 μ L). A mixture containing 5. mu.L of the extract (marker) and a final concentration of 0.5. mu.M of fluorescent probe in a high salinity buffer (s.a.t 100. mu.L) was prepared. A75 μ L sample of this mixture was taken from the cartridge and then incubated at room temperature for 3 minutes. The cassette was emptied and then rinsed 2 times with 75 μ L of high salinity buffer. At the end of the run 75 μ L of high salinity buffer was added. The device was then started using the standard strategy of the Nanogen workstation (registered trade Mark). The reusable cartridge is regenerated.

I.G utilization and interpretation of results

The analysis of three marked products in this first embodiment (see fig. 5) can stimulate especially three possible configurations during the product authentication step.

In the first and second tagged products, the user is able to detect the presence of the encoding polynucleotide. The actual presence of the polynucleotide indicates that the product is likely authentic. With respect to the third product, the complete absence of detection of the encoding polynucleotide indicates that the product is not labeled and, therefore, is not authentic.

After reading the information carried by the encoding polynucleotides present in the first and second products, the user refers to a correlation table (whether the presence of decoy polynucleotides has no effect) indicating the encoding of the encoding polynucleotides (corresponding to the presence of target polynucleotides 1 and 2). These polynucleotides are correctly detected in the first labeling solution, and only these two polynucleotides are detected: the first solution may be authentic. With respect to the second solution, the presence of the unexpected target polynucleotides 3, 4 and 5 makes it impossible to authenticate the product.

Example 2: marking of cosmetic products according to the method of the invention

This example describes the use of several single-stranded deoxyribonucleic acid polynucleotides as labels that are injected into a skin cream and detected according to the technique presented in example 1.

II.A. injection of Polynucleotide into skin cream

Principle of a.1

The marker was first prepared with distilled water without nuclease and then at 10^-12Mol/dm³The concentration of cream caplet (caplet) incorporates the target marker and decoy marker and is 10^-14Mol/dm³The concentration of the cream incorporates a coded marker.

A.2 materials

Synthetic Polynucleotide (Eurofins MWG GmbH), 100 pmol/. mu.L

Cyclic single-stranded deoxyribonucleic acids (coding markers)

Deionization of H₂O, nuclease-free enzyme

Thermal research Fix 2 (registered trademark), VICHY Laboratories

Process of 2.a.3

The premix encoding the target polynucleotide and decoy polynucleotide was injected into 1cm using a pipette P10(Gilson Pipetman, registered trademark)³The resulting final concentration of target and decoy marker was 10 for the Thermal Fix clear (registered trade mark) sample of (1)^-12Molar marker/dm³Final concentration of encoding polynucleotide of 10^-14Mol/dm³. The sample is then kept for its identification.

II.B. extraction and detection of markers

The markers were detected in the same manner as in example 1, except for the polynucleotide extraction step.

B.1 principle of extraction

The marker is extracted from the cream by breaking the emulsion and recovering the aqueous phase. The high temperature (above 80 ℃) is sufficient to reduce the emulsion that makes up the cream and thus to separate the aqueous and lipid phases. Very polar markers are found in the aqueous phase from which they are extracted.

Materials of 2.b.2

-Thermal Fix create (registered trademark) (Vichy) marked

Heating blocks or water baths

Demineralization H₂O, nuclease-free enzyme

Process 2.b.3

The labeled cream was heated at 95 ℃ for 15 minutes and then centrifuged at 10,000rcf for 5 minutes. The aqueous phase is recovered and used to detect the label.

The polynucleotide is then detected according to the method described in example 1.

Example 3: marking spirits according to the method of the invention

This example describes a technique for labelling spirit (spirit) using a target polynucleotide, an encoding polynucleotide and a decoy polynucleotide. The first type of label (target polynucleotide) consists of a pool of 20 single-stranded deoxyribonucleic acids of 20 bases in size. The second marker is a circular single-stranded nucleic acid of 1000 bases in size, the sequence of which contains a study description of the target marker. Thus, the sequence enables to know which of the 20 target markers are meaningful for the coding of the spirit, the other markers being semi-randomly added decoys.

III.A. tags and design of tags

Detailed principles of a.1 labeling technique

The 20 single-stranded DNA markers were generated according to the algorithm shown in the present specification. These markers are different from each other, do not hybridize automatically, and do not readily hybridize to each other. They constitute the target polynucleotide.

The encoding polynucleotide is a 1000 base circular deoxyribonucleic acid. At a given point in time, the sequence of the nucleic acid comprises a cassette containing a combination of target polynucleotides to be retrieved in a product.

FIG. 6 illustrates the principle of the coupling labeling: the circular coded markers contain sites for general information, as well as sites that enable one to know which of the target markers are also present in the mixture and carry the coded information (others are simply decoys).

Thus, a combination of target markers designated only by the coding marker is effective for authentication, and the other markers are merely decoys.

The sequence encoding the marker thus contains a coding cassette, the position of which is hidden. Reading of this sequence enables the user to know which markers are to be found in the library of target markers by cross-checking in the correspondence table.

The combination of target markers enables the identification of the product in a single way: one possible combination is for one possible product. Among the coded markers, downstream of the coding cassette for the combination of target markers to be sought, is a second coding sequence for general information related to the product (e.g., lot, year of manufacture, etc.).

These markers are thus injected into the spirit to be marked. Detection then proceeds in both phases. First, the specific sequence encoding the marker is detected. The first information derived therefrom is as follows:

product authentication. In the case of a counterfeit, there is no coded marker.

Product-wise information: batch N °, manufacturing date, etc.

Unique combinations of target markers to be sought for more thorough detection.

If the first authentication is deemed insufficient, a second authentication is performed on the target marker. This second authentication can infer the following information:

poor forgeries in the absence of target markers or incorrect combinations.

A product from the mixture if a marker is detected in addition to the marker provided by the coded marker.

Obtaining accurate certification of the product by consulting the database.

A.2 materials

Synthetic polynucleotide (Eurofins MWG GmbH), 100 picomoles/μ L.

-et Chandon Champagne (registered trade Mark), Brut Imperial

Cyclic single-stranded deoxyribonucleic acids (coding markers)

Nuclease-free deionized water

A.3 Process

Alpha target marker

The target marker consisted of 20 different polynucleotides obtained by chemical synthesis (Eurofins MWG GmbH). This example was performed using the markings of 5 different products. In each of these products, 20 markers can be used:

primer-1: 5'-AGTCGAGAGCCGATTCCGCT-3' (SEQ ID NO: 25)

Primer-2: 5'-GTCCGAGCAAAGGCTTCCGT-3' (SEQ ID NO: 26)

Primer-3: 5'-AGACCCGTGGGCTCCATTAG-3' (SEQ ID NO: 27)

Primer-4: 5'-CCACCCAGAGGGCTTAGGTT-3' (SEQ ID NO: 28)

Primer-5: 5'-ATCCCACGAGGGTGATCTCG-3' (SEQ ID NO: 29)

Primer-6: 5'-GGAATCCGACCGTGCATGTC-3' (SEQ ID NO: 30)

Primer-7: 5'-CAGAGACGTGACCCGCTGTT-3' (SEQ ID NO: 31)

Primer-8: 5'-GACCCAGGGGTACATTCTCG-3' (SEQ ID NO: 32)

Primer-9: 5'-AAACGAGCCCGTTCCGTGTG-3' (SEQ ID NO: 33)

Primer-10: 5'-GGGAGCCCCAGCATTATCGT-3' (SEQ ID NO: 34)

Primer-11: 5'-GGACGTGAACGCATCCGTCT-3' (SEQ ID NO: 35)

Primer-12: 5'-GGCTGAAGGCCACTACTCTG-3' (SEQ ID NO: 36)

Primer-13: 5'-GTAGGTAGCACACCGTCGCT-3' (SEQ ID NO: 37)

Primer-14: 5'-CAGCCAGGAGATGTCCGTCT-3' (SEQ ID NO: 38)

Primer-15: 5'-GTCCCCAGGTGAGATCATCG-3' (SEQ ID NO: 39)

Primer-16: 5'-CGAGGGACCAGCTTCCGTAT-3' (SEQ ID NO: 40)

Primer-17: 5'-GCCAGTCGCAGGCATGATTC-3' (SEQ ID NO: 41)

Primer-18: 5'-CGCCAGGGTCTCAGTCGTAA-3' (SEQ ID NO: 42)

Primer-19: 5'-GAGCATAGCCGACGTCTTCG-3' (SEQ ID NO: 43)

Primer-20: 5'-GTAGAGTGACACGTCGCTCC-3' (SEQ ID NO: 44)

Of these 20 target markers, only 10 were actually injected into the product. Of these 10 markers, 5 were decoys and 5 were effective markers for the product.Only the sequences encoding the markers will be able to identify which are encoding markers and which are decoys. In addition, the products 1-4 areet sample of Chandon champagne, cuvee impiriale 2005 (registered trademark), product No. 5 iset Chandon champagne, cuv é impp riale 2002 (registered trade Mark).

Table 3.1: labeling products 1-5 with target markers 1-20

	Product 1	Product 2	Product 3	Product 4	Product 5
						Primer 1	-	D	-	L	-
Primer 2	-	D	L	-	-
						Primer 3	-	D	-	-	-
Primer 4	L	L	-	-	L
						Primer 5	-	D	-	L	-
Primer 6	L	L	L	-	L
						Primer 7	-	D	-	-	-
Primer 8	L	L	D	D	L
						Primer 9	-	-	-	L	-
Primer 10	L	L	L	-	L
						Primer 11	-	-	D	D	-
Primer 12	L	L	D	D	L
						Primer 13	-	-	-	L	-
Primer 14	-	-	L	-	-
						Primer 15	D	-	-	-	D
Primer 16	D	-	-	L	D
						Primer 17	D	-	L	-	D
Primer 18	D	-	D	D	D
						Primer 20	D	-	D	D	D

Table 3.1 illustrates the labeling of 5 products. The dashed line indicates no injected marker. The letter L indicates the injected marker and it serves as an identifying feature for the marker. The letter D means that a marker is injected, but it acts as a decoy. Thus, although products 1 and 2 contain different decoys, they contain the same marker. They have the same identifying characteristics. In contrast, products 3 and 4 have the same decoy, but the markers are different. Therefore, they have different identification features. The product 5 has the same identifying characteristics as the product 1. Only the coded marker differs between the two products.

Pre-dilution of the marker to 10 in the marked product^-7Concentration of mols/L (1. mu.L of 10)^-4mol/L of the starting solution diluted in 999. mu.L of the marked product, then 10. mu.L of this intermediate solution diluted in 999. mu.L of the marked product), the marker being injected at a rate of 100. mu.L (using pipette P100), so as to obtain a final concentration of 10 for each of the products 1 to 5^-9moles/L, with a final volume of, for example, 10 mL. The product thus contains 10^-8Molar marker/liter, i.e. 10mL in total^-10Molar markers.

Beta. coded markers

The coded marker synthesized according to the strategy of example 1 was a single-stranded deoxyribonucleic acid with a total length of 1000 bases. The sequence is as follows (SEQ ID NO: 45):

5’ CAGAAGAATGCACGCTCTTTAACGCTTCGCCCTAAAATGGGCCATGACTATTGAGAATACGATACCTTCCCGCGTTAGCATCCCTTCCCTGATGCTGGTA GATCTACACCATCTGTACGGGAGATAAGGCTGGCTGTGCGCTTAGACGGGAACTTGGACCGGAAGAATGCGTACAGCCTTACGCGCATCCGAGTCGTCAC CTACCACACGCTCATGCGCACTTTACGGGTAAAAAGTGTTAATCGTAACAGTGTCGGGACCACTCCTATGCTAATACCAGCGTGGTCCAGTGACGTTTTT LACATAGTAGGTGCTCTAATCTTGCAAACCACCGTTTCATTATCTGTTATTCTCCCTTGCTAATGGCCCGCTCAGCACCGGGTGTTCCCAGAGGAGAGCT

marker 1 encodes a combination of target markers to be searched in a product. Marker 2 encodes general information such as cuvee wine from which the sample was taken. The following (Table 3.2), description of type 1 markers.

Table 3.2: encoding a target marker according to the variable site encoding the marker

TABLE 3.3 encoding the product type according to the second information of the encoded marker

Thus, according to the technique described in example 1, at 10^-11Final concentration of moles/L specific coded markers were injected into the 5 products indicating which coded target authentication markers support the information and indicating from which year champagne (vintage) the sample was taken in this example. They contain the following variable sequences.

Table 3.4: summary of markers for products 1-5

L ═ label

Labeled LP product

III.B. extraction of marker

The detection is performed in a two-step process. The first step consists in detecting the coded marker. Which enables a first authentication of the product by means of the second marker. The first marker is then read to enable the combination of target markers to be searched for to be known.

B.1 principle of extraction

The marker is extracted from its environment (here champagne) and then recovered in an aqueous environment for the purpose of enabling later use of molecular biological identification techniques. The extraction should preferably have a high yield (maximum amount of label recovered, ideally: 100% yield), but it should also free the label from any "contaminating" substances that might interfere with the detection technique. In addition, it is preferred that it should be effective for both types of markers.

Materials of 3.b.2

Ultrapure Phenol (trade Mark) buffer saturated Phenol, Invitrogen

-chloroform/pentanol (24: 1)

Sodium acetate 3M

-95% frozen ethanol (Carlo Erba Rectapur), 75%

Ultrapure glycogen, Invitrogen (trade Mark) 20. mu.g/. mu.mol

Nuclease-free deionized water

Process 3.b.3

The technique used was a technique of extraction using phenol chloroform. For example, the marker was extracted based on 500 μ L of product (champagne).

In a 2mL tube (Eppendorf, registered trademark) containing 500 μ L of the labeled product, 1/2 volumes of phenol (250 μ L) were added, 1/2 volumes of chloroform (250 μ L) were added using a pipette P1000, and then rotated for 10 seconds. Centrifuge at 10,000rcf for 5 minutes.

The aqueous phase was transferred to a new 2mL tube (Eppendorf, registered trade mark) and 1 volume (500 μ L) of chloroform was added. Spin centrifuge at 10,000rcf for 5 minutes. This step was repeated 5 times.

Transfer the aqueous phase to a new tube, then add 3 μ Ι _ glycogen, 1/10 volumes (50 μ Ι _) of 3M sodium acetate and 2.5 volumes (1250 μ Ι _) of frozen ethanol. Mixing and then incubation at-20 ℃ for 1 hour.

Centrifugation at 10,000rcf for 10 minutes. The supernatant was discarded and the pellet was carefully washed with 200 μ L of 75% frozen ethanol. The precipitate is then dried. This was placed in 20. mu.L of nuclease-free deionized water.

Detection of III.C. coded markers

Principle of 3.c.1

The encoded markers are detected by chain polymerization techniques. Two primers are required for this purpose. The first type of primer is complementary to a region located 5' to variable sequence 1. This primer is referred to as a Universal (Universal) primer because it does not depend on the variability of the target markers (it recognizes a site common to all these markers). As for the second primer, it is complementary to variable site 2. Thus, the same number of primer pairs (here, two pairs) as the type of variable sequence 2 are used.

FIG. 7 shows the primary labeling of the encoded markers by PCR detection. The figure continues with the use of these two types of primers, the template, and the strand that is complementary to itself and that is generated during each first PCR cycle.

For each type of variable sequence 2, the markers extracted from the product were subjected to a polymerization chain reaction. Specific amplification of the encoded marker for a pair of primers reveals the presence of type 2 sequences. The absence of amplification indicates an unlabeled product and thus may be a counterfeit. Amplification of the marker using the wrong primer pair, or amplification of the marker using several primer pairs revealed falsification of the product (falsification of la cuvee, mixture, etc.).

Material 3.c.2

Taq polymerase (Applied Biosystems) AmpliTaq Gold

10 buffer XPCR buffer II (applied biosystems)

-MgCl₂Solution (25mM) (Applied Biosystems)

Universal sense primer (Eurofins MWG GmbH), 100 pmol/. mu.L

5’ACGTTGCAGCGAGCG 3’(SEQ ID NO：56)

Antisense primers (see variable sequence V2: antisense primers are the reverse complement thereof) (Eurofins MWG GmbH, 100 pmol/. mu.L)

-dNTP(2mM)

Agarose gel (agarose electrophoresis grade, Invitrogen, number: 15510-

Tris-borate buffer EDTA (TBE)0.5 in volume

Ethidium bromide

Method of 3.c.3

Reagents were added to a 200 μ L test tube (Eppendorf, registered trademark) using pipettes P2, P10, and P100 according to table 3.5.

Table 3.5: polymerization chain reaction

Reagent	BodyProduct (mu L)
		Template: extracting the product	5
General sense primer (10pM)	0.5

Specific antisense primer (10pM)	0.5
		Buffer 10X	5
dNTP(2mM)	2.5
		MgCL₂(25mM)	2.5
AmpliTaq Gold(5U/μL)	0.5
		H₂O q.s 50μL	33.5

Therefore, the polymerization chain reaction was performed on a GeneAmp PCR System 9700 apparatus (Applied biosystems) using the following cycle:

-94℃ 5’

-94℃ 30”|

30 "| > 40 cycles at-55 ℃ |

-72℃ 30”|

-72℃ 7’

Thus, the PCR products were deposited on a 0.5% agarose gel prepared in TBE 0.5 Xand allowed to migrate to 10V cm in buffer TBE 0.5X^-1. After sufficient migration time, the gel was placed in a bath containing the BET, rinsed and then developed under UV light. For each product, the absence of a band revealed no detectable marker, or sequence 2 did not correspond to the type of primer used. The band (size 358bp) corresponds to the amplification and therefore to the detection of the specific sequence encoding the marker.

For each positive detection, the amplicon is retained for possible detection of the target marker.

Detection of target markers

Principle of d.1

Target markers are searched in products for more thorough detection of the markers. The target marker can be detected only by reading the encoded marker. The detection is performed by a polymerase chain reaction on the amplicons obtained previously. The first step is to read the information contained on the coded marker and thus on the amplicon. This information (sequence 1) makes it possible to know, by means of a correlation table, which target markers are present in the product whose presence carries the marker information. By knowing this, detection of all target markers is achieved by DNA microarrays. After revealing the test, the user is able to know which target markers are present or absent in the product and can compare these results with results theoretically obtained by reading the information carried on the coded markers.

D.2 materials

Universal sense primer (Eurofins MWG GmbH), 100 pmol/. mu.L

5’ACGTTGCAGCGAGCG 3’(SEQ ID NO：56)

Big Dye (registered trademark) terminator V3.1(Applied Biosystems)

-a template: coding amplicon markers

Nuclease-free deionization H₂O。

D.3 Process

Each amplicon was subjected to a sequencing reaction in a 200. mu.L tube (Eppendorf, registered trade Mark) according to the strategy summarized in Table 3.6.

Table 3.6: sequencing reactions

Reagent	1 sequence
		Big Dye (registered trademark)	2μL
Amplicon (template)	5μL
		Primer 10pM	1.6μL
H₂O	sat.10μL

The reaction was then run on a GeneAmp PCR System 9700 apparatus (Applied biosystems) using the following cycle:

-96℃ 1’

-96℃ 10”|

-50 ℃ 5 "| > 25 cycles

-60℃ 4’ |

After column-based purification (Qiagen, registered trade mark), the reactions were sequenced on a 16 capillary sequencer ABI3100(Applied Biosystem).

From the results of these sequences, the variable sequences (or markers 1) were extracted. These sequences enable one to find out by consulting the following table (table 3.7) which target markers have to be present in the mixture:

table 3.7: encoding a target marker according to the variable site encoding the marker

The target markers were then detected according to the techniques summarized in example 1. This test reveals how relevant information is drawn by comparison with the coded marker, although about 10 markers are present in each product.

Example 4: marking pharmaceutical products according to the method of the invention

This example shows how a pharmaceutical product in the form of a caplet (e.g. a form used for many medical preparations) can be marked according to the marking method of the present invention. The strategy of example 1 was used.

Iv.a. injection of polynucleotides into capsules

Principle of a.1

The marker was first placed in an ethanol solution (80%) and then 10% during the caplet manufacturing process^-12At a final concentration of Mole/400 mg caplet target and decoy markers were introduced at 10^-14Mols/caplet concentration incorporates the coded marker. For this example, the caplet does not contain any active ingredient.

A.2 materials

Synthetic Polynucleotide (Eurofins MWG GmbH), 100 pmol/. mu.L

Cyclic single-stranded deoxyribonucleic acids (coding markers)

Nuclease-free deionization H₂O

Beta-lactose, Sigma Aldrich (registered trademark) # L3750-500G

Corn starch, Sigma Aldrich (registered trademark) # S4180-500G

Monocalcium phosphate, Sigma Aldrich (registered trademark) #307645

-syrup

Magnesium stearate, Sigma Aldrich (registered trademark) #26454-1KG

A.3 Process

a. Preparation of marker solutions

The marker was prepared in the same manner as in example 1.

Placing the premix in an 80% ethanol solution to introduce the polynucleotide into the caplets is accomplished in any manner.

b. Preparation of caplets

Caplets were prepared from 300g pellets. The different powders constituting the pellets were first weighed and then mixed according to the following formulation ("Lodigge" type cylindrical high speed granulator):

-beta-lactose: 120g of

-corn starch: 60g of

Calcium dihydrogen phosphate: 120g of

A wetting solution was then prepared from 100g of syrup and 750. mu.L of the marker solution. The wetting solution is then gradually added to the mixture of powders until a wet mass is obtained with the appearance of coarse wheat flour, and the previous mixture is then granulated, obtaining a wet vermicular granulate (using a vibrating granulator).

The pellets are then dried at 60 ℃ until a moisture of 4-6% is obtained, and then sieved in a sieve column to remove fine particles. After lubrication with 1% magnesium stearate, the pellets were loaded into a press and then compressed.

IV.B. extraction and detection of markers

B.1 principle of extraction

The marker is extracted by grinding the caplets and then recovering the polynucleotide in the aqueous phase.

B.2 materials

Marked caplets

Heating blocks or water baths

Demineralization H₂O, nuclease-free enzyme

B.3 Process

The caplets were ground using a mortar and pestle to obtain a very fine powder. This powder was then mixed with 1mL of distilled water in a 1.5mL test tube (Eppendorf, registered trademark). After heating at 70 ℃ for 15 minutes, the tube was centrifuged at 5000rcf for 5 minutes to remove solid particles. The aqueous phase was placed in a new tube for polynucleotide authentication steps. The polynucleotide can then be detected according to the method described in example 1.

Example 5: marking of food products according to the method of the invention

This example shows how food products such as pizza dough can be marked and markers extracted therefrom. The marker was injected into fresh dough during preparation. The marker may thereafter be detected in the finished product, either cooked or uncooked, ready for consumption.

Marking of V.A dough

Principle of a.1

The markers were pre-diluted in demineralised water. A mixture of target, encoding and decoy polynucleotides is used as shown in example 1. They are then introduced into the pizza dough recipe, which is then cooked.

A.2 materials

Synthetic Polynucleotide (Eurofins MWG GmbH), 100 pmol/. mu.L

Cyclic single-stranded deoxyribonucleic acids (coding markers)

Nuclease-free deionization H₂O

-pizza dough ingredients: t45 flour "general purpose wheat for savoury products" Francine (registered trademark), olive oil "ultrapure cold pressed olive oil" Carapelli (registered trademark), yeast "baker's yeast" Francine (registered trademark), sugar "white sugar" Daddy (registered trademark), salt "essential white iodized salt" crebos (registered trademark).

A.3 Process

The preliminary solutions of target marker, coding marker and decoy marker prepared as summarized in example 1 were added and added to the pizza dough during its manufacture using a pipette P200(Gilson Pipetman, registered trademark) in an amount sufficient to obtain the final concentrations described belowDough to obtain a final concentration of marker of 1^E10 moles/kg (for each non-decoy marker, i.e. 2)^E-9 moles/kg polynucleotide). The following table (table 5.1) indicates the amounts of ingredients used to obtain 828 grams of pizza dough.

Table 5.1: making marked pizza dough

Composition (I)	Amount or volume
		Flour	500g
Olive oil	50g
		Yeast	12g
Candy	8g
		Salt (salt)	8g
Water (W)	250mL
		Preliminary solution for polynucleotides	166μL

The dough was then baked in an oven for 15 minutes at an average temperature of 240 ℃.

Extraction of v.b. marker

Extraction of the marker by dissolving a part of the cooked dough in demineralized water: 1g of cooked dough was reduced to a powder and then placed in 10mL of water. The entire mixture was then mixed vigorously and subsequently heated at 94 ℃ for 15 minutes. After centrifugation at 10,000rcf for 5 minutes, the aqueous phase was recovered and stored at 4 ℃ for detection of the label. These markers were detected according to the technique shown in example 1.

Example 6: marking tobacco articles according to the method of the invention

This example shows how tobacco articles are marked. Tobacco products are labeled by direct absorption of the tobacco product using polynucleotides such as those shown in example 1.

VI.A labeled tobacco products

Principle of a.1

The marking relates to tobacco articles known as "blondes" (Virginia cigarettes). The sample is allowed to absorb a mixture of target, coding and decoy polynucleotides such as described in example 1.

A.2 materials

Synthetic Polynucleotide (Eurofins MWG GmbH), 100 pmol/. mu.L

Cyclic single-stranded deoxyribonucleic acids (coding markers)

-ethanol (denatured absolute ethanol, Sigma-Aldrich #676829)

Nuclease-free deionization H₂O

Marlboro cigarette (registered trademark), Philip Morris Products Plc.

A.3 Process

Tobacco products were labeled with a polynucleotide solution prepared according to the method described in example 1.

1 gram of cigarette extract was labelled to finally obtain 10^-12Molar marker per gram of tobacco product: a sufficient amount of the pre-marking solution was poured onto the tobacco product using a pipette P200(Gilson Pipetman, registered trade Mark), and the whole was then vigorously mixed for 15 minutes at 37 ℃.

The tobacco product thus marked is then kept at a temperature of 16 ℃ and a humidity of 70%.

VI.B marker extraction and identification

Extracting the marker by immersing the marked tobacco product in demineralized water: 1 gram of the tobacco product was immersed in 10mL of deionized distilled water and then heated at 94 ℃ for 15 minutes.

After centrifugation at 10,000rcf for 5 minutes, the aqueous phase was recovered and stored at 4 ℃ for detection of the label. These markers were detected according to the technique shown in example 1.

Example 7: marking hydrocarbons according to the method of the invention

This example shows how hydrocarbons (crude oil) are labeled and which technique can be used to extract the markers and detect the markers.

VII.A labeling of hydrocarbons

Principle of a.1

In this example, crude oil samples were labeled with polynucleotides previously prepared in polar organic solvents. The tag consists of the target, encoding and decoy polynucleotides as described in example 1.

A.2 materials

Synthetic Polynucleotide (Eurofins MWG GmbH), 100 pmol/. mu.L

Cyclic single-stranded deoxyribonucleic acids (coding markers)

Nuclease-free deionization H₂O

Dimethyl sulfoxide (DMSO), Euromedex ref UD8050-A

-crude oil (IFP)

A.3 Process

Solutions of single stranded target polynucleotides, encoding polynucleotides and decoy polynucleotides were prepared as described in example 1.

A second preliminary mixing was then achieved in DMSO prior to inserting the marker into the crude oil.

The second preliminary solution of marker was then mixed with the crude oil using a precision pipette (Gilson Pipetman, registered trade Mark) in an amount sufficient to obtain a final concentration of 10^-10molar/L of label.

The labeled crude oil is then stored at room temperature for use in identifying the marker.

II.B. extraction and detection of markers

Principle of b.1

The marker is extracted from its non-polar environment and then recovered in an aqueous environment for later use in identification techniques.

B.2 materials

Ultrapure Phenol (trade Mark) buffer saturated Phenol, Invitrogen.

-chloroform/pentanol (24: 1)

-3M sodium acetate

-95% frozen ethanol (Carlo Erba Rectapur), 75%

Nuclease-free deionized water

Hexane (Sigma, ref H9379-1L)

Process 7.b.3

The technique used is a phenol-chloroform extraction technique. For this example, the marker was extracted from 500. mu.L of crude oil.

Add 2 volumes of hexane.

Add 1/2 volumes of phenol (250 μ L), 1/2 volumes of chloroform (250 μ L) and 1/2 volumes of water (250 μ L) and mix very vigorously for 30 seconds. Centrifuge at 10000 rcf for 30 min.

Transfer the aqueous phase to a new tube and add 1 volume of chloroform. Mix very vigorously and centrifuge at 10,000rcf for 20 minutes. This step was repeated 5 times.

Transfer the aqueous phase to a new tube and add 1/10 volumes of 3M sodium acetate and 2.5 volumes of frozen ethanol. Mixing and then incubation at-20 ℃ for 1 hour.

Centrifugation at 10,000rcf for 10 minutes. The supernatant was discarded and carefully washed with 200. mu.L of nuclease-free deionized water.

These markers can be detected according to the technique shown in example 1.

Example 8: marking of fresh food products according to the method of the invention

In this example, a mixture of polynucleotides is used to label fresh food. The product is a dairy product: yogurt.

VIII.A labeled yogurt

Principle of a.1

The markers were pre-diluted in demineralised water. The tag consists of the target polynucleotide, coding nucleotides and decoy nucleotides as described in example 1.

A.2 materials

Synthetic Polynucleotide (Eurofins MWG GmbH), 100 pmol/. mu.L

Cyclic single-stranded deoxyribonucleic acids (coding markers)

Nuclease-free deionization H₂O

Activia yogurt (registered trademark) manufactured by Danone (registered trademark)

A.3 Process

A pipette P10(Gilson Pipetman, registered trade Mark) was used to add a preliminary solution of the target polynucleotide, encoding polynucleotide and decoy polynucleotide to the yoghurt in an amount sufficient to obtain a final concentration of 1^E10 moles/kg of marker (for each non-decoy marker, i.e. 2)^E9 moles/kg polynucleotide) and then 2 μ L of the preliminary solution was injected into the bulk product. Then, the whole was homogenized well by using a sterile spatula (spatula).

The labelled yoghurt was then stored at 4 ℃ while waiting for the marker to be identified.

III.B. extraction of marker

The markers were extracted by dissolving part of the yogurt in demineralised water: 1 gram of yogurt was taken in 10mL of water. The whole was mixed vigorously and then heated at 94 ℃ for 15 minutes. After centrifugation at 10,000rcf for 5 minutes, the aqueous phase was recovered and stored at 4 ℃ for detection of the label. These markers can be detected according to the technique shown in example 1.

Example 9: marking beverages according to the method of the invention

The non-alcoholic soft drinks were labelled using polynucleotides such as described in the examples: orangina (registered trademark), Schweppes International Limited.

IX.A labeled beverage

Principle of 9.a.1

Target markers, coding markers and decoy markers were pre-diluted in demineralised water. They are then mixed directly into the beverage.

A.2 materials

Synthetic Polynucleotide (Eurofins MWG GmbH), 100 pmol/. mu.L

Cyclic single-stranded deoxyribonucleic acids (coding markers)

Nuclease-free deionization H₂O

Orangina (registered trade Mark), Schweppes International Limited.

A.3 Process

Pre-mixing is achieved in deionized water prior to insertion of the marker into the beverage.

To obtain a final concentration of 1^E10 moles/L of marker (for each non-decoy polynucleotide, i.e. 2)^E-9 moles/kg polynucleotide) of the marker is mixed with the beverage.

The marked beverage was then stored in a sealed container at 4 ℃.

IX.B. extraction of marker

The extraction of the marker was performed using the phenol-chloroform technique shown in example 1.

These markers can be detected according to the technique shown in example 1.

Example 10: marking a paper substrate according to the method of the invention

This example shows how the paper base is marked (paper base) and then how the marker is extracted from the paper base. The label is absorbed directly through the paper. The marker is then detected by dissolving the marked paper.

X.A Label of paper

Principle of a.1

The target, coding and decoy polynucleotides as described in example 1 were pre-diluted in demineralised water. Thereby absorbing the droplets onto the surface of the paper.

A.2 materials

Synthetic Polynucleotide (Eurofins MWG GmbH), 100 pmol/. mu.L

Cyclic single-stranded deoxyribonucleic acids (coding markers)

Nuclease-free deionization H₂O

Paper tray "1 QUALITATIVE Filter Paper", Whatman (registered trademark).

A.3 Process

Pre-mixing as summarized in example 1 was performed in deionized water prior to depositing the marker on the paper.

Thus a preliminary solution of the marker was deposited on the paper to deposit 1E-12 moles of marker. 1 μ L of the preliminary solution was deposited using a precision pipette P10(Gilson Pipetman, registered trade Mark), thus resulting in the formation of a 5 mm-diameter disc.

The paper is dried in the open air and then held for detection of the marker.

Extraction of the marker

The markers were extracted by dissolving part of the paper in demineralised water: mixing 1cm²The paper containing the marker disks was cut into small pieces and then 10mL of water was added. The whole was then heated at 94 ℃ for 15 minutes.

The pulp was mixed very vigorously and then homogenized by sucking/discharging with a pipette. After centrifugation at 2000rcf for 5 minutes, the aqueous phase was recovered and stored at 4 ℃ for detection of the label. These markers were detected according to the technique shown in example 1.

Example 11: olfaction and ageing tests on the marked products obtained according to the method of the invention

The stability test and the aging test over time were carried out jointly by the cosmetic industry. Olfactory tests can check that the addition of target, encoding and decoy polynucleotides does not alter the physicochemical and olfactory properties of the perfume by labeling according to the invention. These tests have been carried out on several perfumes and according to several conditions: at 5 ℃ for one month (used as control), at 50 ℃ for one month (stimulating accelerated ageing of the perfume) and exposure to daylight for one month.

After aging, tests have shown that, whatever the aging method used, the solutions containing the polynucleotides retain the same physicochemical and olfactory properties as the solutions not containing the polynucleotides. In addition, for each vial, the marker has been extracted and then successfully identified.

Example 12: olfaction and ageing tests on the marked products obtained according to the method of the invention

Fig. 8 shows a detection scheme for detecting the marking of the present invention and the authentication of the marked product of the present invention.

In this figure:

1. identifying the encoded marker. The first product authentication level is in the presence of a coded marker. If the product does not contain a coded marker, the product is a counterfeit.

2. Decoy markers and target markers are then identified in the product. On this figure, the product is labeled with a series of 20 putative target polynucleotides and decoy polynucleotides. Therefore, it contains 1 to 20 markers selected from the batch. In this step of the method, the target marker and the decoy marker are not distinguished (it is not possible to tell whether a polynucleotide is the target polynucleotide or the decoy polynucleotide).

3. The properties of the encoding polynucleotide (here polynucleotide a) detected during the first step are then sent to the label provider. With this information, the tag provider recovers the tagged decryption key from the database, thus allowing him/her to distinguish decoy polynucleotides from the series of decoy and target polynucleotides.

4. Using the decryption key, the user is only interested in the target polynucleotide. The user can thus read the code consisting of the presence or absence of the target polynucleotide (in this example, the codes are-, + and-).

5. Reading of the code thus enables the user to check, using the secure database, whether the authentication code does indeed correspond to the product of interest. In the opposite case, it may be an illegal copy, a mixture of products or a product abuse.

Reference to the literature

[1] Molecular Cloning, Maniatis, Cold Spring-Harbor, second edition, pages C3-C14.

[2]Smith TF，Waterman MS(1981).Identification of common molecular subsequences，J Mol Biol 1981 Mar 25；147(1)：195-7.

[3]Xin W，Zhang YM，Xiao JH，Huang DW(2003).Construction of linear functional expression elements with DNA fragments created by site-specific DNA nickase，N.Bpu10I，and exonuclease III，Biotechnol Lett.2003 Nov；25(22)：1913-6.

[4]Caruthers MH，Beaucage SL，Efcavitch JW，Fisher EF，Matteucci MD，Stabinsky Y.，(1980)New chemical methods for synthesizing polynucléotides，Nucleic Acids Symp Ser.1980；(7)：215-23.

[5]Wheeler，C.J.，L Sukhu，G.Yang，Y.Tsai，C.Bustamente，P.Felgner，J.Norman，M.Manthorpe.(1996).Converting an alcohol to an amine in a cationic lipid dramatically alters the co-lipid requirement，cellular transfection activity and the ultrastructure of DNA-cytofectin complexes.Bioch.Biophys.Acta 1280：1.

[6]Felgner，J.H.，R.Kumar，C.N.Sridhar，C.J.Wheeler，Y.S.Tsai，R.Border，P.Ramsey，M.Martin，P.L.Felgner.(1994).Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations.J.Biol.Chem.269：2550

[7]Ogris M，Steinlein P，Carotta S，Brunner S，Wagner E(2001)NA/polyethylenimine transfection particles：influence of ligands，polymer size，and PEGylation on internalization and gene expression，AAPS PharmSci.2001；3(3)：E21.

[8] Molecular cloning, Maniatis, CoId Spring-Harbor, second edition, pages E3-E4.

[9] Molecular Cloning, Maniatis, CoId Spring-Harbor, second edition, pages 9.47-9.57.

[10]Kabilov MR，Pyshnyi DV，Dymshits GM，Gashnikova NM，Pokrovskii AG，Zarytova VF，Ivanova EM (2002).A new approach to detect a particular DNA sequence by UV-immobilization of its hybridization complex with a highly specific probe resulting from ligation of a tandem of short oligonucleotides in solution，Mol Biol (Mosk).2002 May-Jun；36(3)：424-31.

[11]Ramsay G(1998).DNA chips：state-of-the art.Nat Biotechnol.1998 Jan；16(1)：40-4.

[12] Ivanovskaia MG, Kozlov IA, Lebedeva IV, Shabarova ZA (1994). A new method of evaluating immibilisation of oligooxyducucides on nylon membranes for hybridization with nucleic acids, Mol Biol (Mosk).19949 months-10 months; 28(5): 1176-82.

Claims

1.A method of product labeling, the method comprising the step of adding to or in the product a plurality of single stranded polynucleotides, said plurality of polynucleotides comprising:

-at least one target polynucleotide consisting of a single-stranded polynucleotide having a predetermined length and sequence, and

-decoy polynucleotides of the same or different predetermined length and of the same or different predetermined sequence, the length of the decoy polynucleotides being the same or different from the at least one target polynucleotide and the sequence of the decoy polynucleotides being different from the sequence of the at least one target polynucleotide,

wherein each of the target polynucleotide and decoy polynucleotide does not hybridize to any other polynucleotide of the plurality of polynucleotides, and

wherein a polynucleotide of the plurality of polynucleotides is a deoxyribonucleic acid or ribonucleic acid sequence comprising the same ratio of four natural or modified bases A, C, G and T, or A, C, G and U, respectively.

2. The method of claim 1, wherein the plurality of polynucleotides further comprises at least one identification polynucleotide consisting of a single-stranded polynucleotide having a predetermined length and sequence for identifying the nature and sequence of the at least one target polynucleotide, wherein each of the identification polynucleotides does not hybridize to any other polynucleotide of the plurality of polynucleotides.

3. The labeling method of claim 1, wherein said polynucleotides in said plurality of polynucleotides are circular or linear.

4. The labeling method of claim 1, wherein at least two target polynucleotides are used, one being a circular polynucleotide and the other being a linear polynucleotide.

5. The labeling method of claim 3 or 4, wherein the linear polynucleotides comprise a variable end that is variable between different polynucleotides and a constant end that is constant between different polynucleotides.

6. The labeling method of any preceding claim, wherein said at least one target polynucleotide in said plurality of polynucleotides is 5 to 50 nucleotides in length.

7. The labeling method of any one of the preceding claims, wherein said polynucleotides in said plurality of polynucleotides are 5 to 5000 nucleotides in length.

8.A labelling method according to any preceding claim, wherein the adding step is carried out by adding the plurality of polynucleotides into or onto an end product during manufacture of the product.

9.A labelling method according to any of claims 1 to 8, wherein the adding step is carried out by adding the plurality of polynucleotides to the surface of the product.

10. The labeling method of any one of the preceding claims, wherein the step of encapsulating the plurality of polynucleotides in a lipid carrier is performed prior to the step of adding.

11. The labeling method of any preceding claim, wherein the introduction of the plurality of polynucleotides is performed on or in a component of the product.

12. A labelling method according to any preceding claim, wherein the concentration of the plurality of polynucleotides in the product following addition of the plurality of polynucleotides is 10^-6Mol/dm³～10^-18Mol/dm³。

13. A marked product obtainable by the method of any one of claims 1 to 12.

14. The marking product of claim 13, selected from the group comprising fragrances, cosmetics, sanitary products, food products, condiments, botanical extracts, tobacco products, beverages, textiles, leather products, pharmaceuticals, powders, varnishes, inks, food products, hydrocarbons, paper, coatings, and chemical products and compounds.

15. A method for the detection of a marker of a product according to claim 13, said method comprising a step of analyzing said plurality of polynucleotides, said analyzing step enabling in particular the detection of said at least one target polynucleotide, said method comprising the following successive steps:

(i) contacting said plurality of polynucleotides with a solid phase substrate having probe sequences immobilized thereon, said probe sequences being complementary to one of said ends of said at least one target polynucleotide of said plurality of product-tagged polynucleotides, said contacting enabling immobilization of a target polynucleotide on said substrate by hybridization to said complementary probe sequences immobilized on said substrate;

(ii) (ii) removing polynucleotides not hybridised by step (i);

(iii) detecting the presence of a target polynucleotide on the substrate; and

(iv) (iv) comparing the result of step (iii) with the contents of a database capable of authenticating and certifying said product.

16. The method of claim 15, further comprising, prior to the analyzing step, the steps of:

(a) taking a sample of the product; and

(b) extracting said plurality of polynucleotides from said sample, and performing said analyzing step on said plurality of polynucleotides extracted from said sample.

17. The assay of claim 15 or 16 wherein the step of assaying comprises performing a polymerase chain reaction of the target polynucleotide.

18. The assay of any one of claims 15-17 wherein the analyzing step is performed by immunoassay.

19. The label detection method according to claim 15, wherein a polynucleotide complementary to the target polynucleotide is immobilized on the solid phase substrate using biotin/streptavidin linkage.

20. The label detection method of claim 15, wherein the polynucleotide complementary to the target polynucleotide is immobilized on the substrate by forming a covalent bond with an uncharged nylon membrane, which forms the solid phase substrate.

21. The label detection method as claimed in any one of claims 15 to 20, wherein the target polynucleotide immobilized on the substrate is detected using a polynucleotide labeled with a labeling agent selected from the group consisting of fluorescein, colloidal gold particles and an enzyme and complementary to the other end of the target polynucleotide.

22. The indicia detection method of claim 15, wherein the database is capable of determining the source of the product.

23. A method of signature detection as claimed in claims 15 to 22 wherein the database is capable of identifying counterfeits of genuine products.

24. The label detection method as claimed in any one of claims 15 to 23, wherein the extraction defined in step (b) is a phenol-chloroform extraction.

25. The detection method according to any one of claims 15 to 24, wherein when the target polynucleotide is a ribonucleic acid, the analyzing step comprises reverse transcription of the ribonucleic acid into a deoxyribonucleic acid.

26. The detection method of any one of claims 15 to 24, wherein the analysis step comprises a step of sequencing the target polynucleotide.