DE102005046361A1 - Method for the differentiation of Salmonella serovars - Google Patents

Abstract

The The invention relates to a method for laboratory diagnostic detection and for distinguishing Salmonella serovars, especially for differentiation of Salmonella enterica subsp. Serovar Parathyphi B and Salmonella enterica subsp. enterica serovar Java using oligonucleotide primers and Oligonucleotide probes. Furthermore, the invention relates to oligonucleotides, a biochip and a kit for use in this procedure.

Description

The The invention relates to a method for laboratory diagnostic detection and for the distinction of Salmonella serovars, in particular for Distinction of Salmonella enterica subsp. enterica serovar parathyphi B and Salmonella enterica subsp. Enterica Serovar Java using of oligonucleotide primers and oligonucleotide probes. Further concerns the invention uses oligonucleotides, a biochip and a kit in this procedure.

Salmonella enterica subspec. enterica is one of 6 subspecies of the species Salmonella enterica, which is among the species with the greatest diversity in the kingdom belongs to the bacteria. There are also two pathogenic Salmonella enterica subspec. Enterica groups differed for infections of humans responsible are: typhoid Salmonella species such as S. enterica subspec. enterica serovar typhi and Paratyphi A, B and C lead to systemic diseases such. B. sepsis and organ manifestations (typhoid fever) while the enteric Salmonella species (eg Salmonella enterica subspec. Enterica Serovar Java) cause diarrhea and epidemiological have their origin in animal breeding. In salmonella diagnostics is the distinction of enteric and typhoid Salmonella from medical and food law. For employees in the food sector the evidence has typhoid Salmonella serious consequences (inter alia, an activity ban until detection of 10 salmonella-free stool samples). Usually can by agglutination (Gruber reaction), ie by the aggregation of pathogens by antisera, about 2,200 serovars of Salmonella enterica subspec. enterica according to the Kaufmann-White scheme accordingly the antigenic structure of cell wall polysaccharides or O antigens (somatic antigens) and the flagellum antigen or H antigens (from protein, flagellin) (Popoff, M.Y. 2001. Antigenic formulas of Salmonella serovars. W. H. O. Collaborating Center for Reference and Research on Salmonella. Institut Pasteur, Paris, France). The serovars are then usually either enteric or typhoid Assign Salmonella. An exception are the serovars Salmonella Paratyphi B (typhoid Salmonella) and Salmonella java (enteric salmonella). Own this in the Kaufmann-White scheme the same antigenic formula, which is why Salmonella Java is often not considered own serovar, but the serovar Salmonella Paratyphi B is attributed.

This at the same time means that the distinction of these two serovars in the samples to be examined alone on the basis of their antigenicity possible is and other phenotypic or genotypic-differentiable characteristics should be analyzed.

In view of this problem, a method was developed by Kristensen and Kaufmann (Kristensen M. and Kaufmann F., 1937. Bacteriological and Clinical Experiences on Infections with D-tartaric Acid-Beating Paratyphoid B-Bacilli, Z. Hyg. Infektionskr. 120: 149-154), that the differentiation of L (+) - tartrate- so D-tartrate-fermenting Salmonella enterica subsp. enterica isolates based on phenotypic properties. This procedure was later modified by Alfredsson and coworkers (Alfredsson GA, Barker RM, Old DC and Duguid JP, 1972. Use of d-tartaric acid isomers and citric acid in the biotyping of Salmonella typhimurium, J. Hyg. 70: 651-666 ). The assay detects Salmonella Java as having the ability to ferment to D-tartrate, whereas Salmonella Paratyphi B does not utilize the named substrate. For this purpose, 8 ml of an autoclaved D-tartate-containing nutrient solution (in 1 L of water are dissolved 10 g of peptone, 1% potassium sodium tartrate tetrahydrate, pH 7.4, and 0.0023% bromothymol blue sodium salt) in one Test tube after inoculation with 50 .mu.l of a saline bacterial suspension having a density of 10 ⁹ bacteria per ml, incubated standing at 37 ° C for 3 and 6 days standing. Thereafter, the cultures are assayed by adding 0.8 ml of saturated aqueous lead acetate solution to the D-tartrate reaction. The resulting precipitate is homogenized by shaking. D-tartrate fermenting strains are identified by the formation of a small amount of precipitate 1 to 2 hours after addition of lead acetate. In contrast, the presence of a flocculent fine precipitate greater than half the total volume indicates strains that can not ferment D-tartrate.

Although this method has long been used as a standard method in World Health Organization laboratories, the high cost of time is a serious drawback of this method and the result of "D-tartrate negative" can only be established after up to 10 days. that the accuracy of the so-called lead acetate test depends on different parameters such as the CO ₂ content of the gas atmosphere in the culture and the density of the inoculum, so that the cultivation-dependent method provides reaction results that are not always clear.

One faster PCR-based detection method of D-tartrate fermentation was developed by Malorny and co-workers (Malorny B., Bunge C. and Helmut R., 2003. Discrimination of d-tartrate-fermenting and non-fermenting Salmonella enterica subsp. enterica isolates by genotypic and phenotypic methods. J. Clin. Microbiol. 41: 4292-4297). In this The amplicons are then amplified as bands in a subsequent procedure Gel electrophoresis using molecular size standards demonstrated. The identification of typhoid Salmonella can by means of this PCR method already present after a day and provides therefore an improvement. However, a disadvantage of this method is that the actual detection of the pathogenic serovars on the Non-emergence of a PCR product supports. As the expert sufficient is known, the non-emergence of a PCR product several Causes have: Forget one of the two primers the PCR mixture add, or if one sets too high or too low a concentration of salts or DNA, this may be amplification in a non-sequence dependent manner prevent. Furthermore the PCR product must be separated in a DNA gel and atrophy stained the gel and these treatment steps can thus both a longer time claim, as well as an increased risk of contamination (danger the carryover of PCR products in later reactions) over modern ones Represent real-time PCR procedures. Another disadvantage of this method is the lack of DNA sequence-dependent confirmation of the identity of the PCR product z. B. by using suitable restriction enzymes (RFLP), by Using specific probes or by sequencing the PCR product. The sole proof of a PCR product or its Non-emergence generally no longer corresponds to what is to be demanded State of the art for diagnostic purposes.

In a scientific treatise on molecular properties of systemic and enteric pathovars of Salmonella enterica (Prague R., Rabsch W., Streckel W., Voigt W., Tietze and Tschäpe H., 2003. Molecular properties of Salmonella enterica serotype paratyphi B: distiguish between its systemic and its enteric pathovars. J. Clin. Microbiol. 41: 4270-4278) are u. a. polymorphic genes for differentiation listed. The genome typhoid Pathovare is therefore generally by the presence of a bacteriophage (ΦSopE309) whose gene is sopE1 in combination with consideration a second gene avrA as an identification feature compared to the Enteric serovars can be used. Typhoid serovars have the sopE1-containing bacteriophage but not the avrA gene in their genome, whereas enteric serovars are heterogeneous in this respect occur: you can either only the avrA gene or the avrA gene in combination with the sopE1 gene or neither gene. For a detection method of Gene sopE1 and avrA are missing by PCR as a diagnostic tool the description of a possibility for one DNA sequence-dependent confirmation the identity the PCR products of said genes using DNA probes. Staining the PCR products brings the above-mentioned disadvantages. Another, already mentioned above disadvantage of this method The prior art is that for determining the molecular weight the PCR products a time-consuming electrophoretic separation of PCR products is required.

task It is therefore an object of the present invention to provide a method with a fast DNA sequence-specific differentiation of Salmonella Paratyphi B and Salmonella Java is possible. More specifically is the object of the invention underlying the preferred provision of several, with pathogenicity the pathogen correlating factors in a single procedure. Furthermore, it is desirable both the specific oligonucleotides, ie primers and probes, in multiplex-compatible form as well as to provide a kit, with which the amplification of the PCR products and the detection in a single pass is easily possible.

These Problems are solved by the subject matters of the independent claims.

advantageous Embodiments are described in the subclaims.

in the Following are some terms explained for the description of the present invention.

Under Serovar generally becomes a serological variant of a species (Species) or subspecies understood by their antigenic properties is characterized.

In the context of the present invention, an oligonucleotide is understood to mean an isolated and purified molecule, which as a rule consists of not less than 10 and preferably not more than 100 nucleotides exists. Oligonucleotides are used within the process as oligonucleotide primers or oligonucleotide probes. The oligonucleotides according to the invention may be molecules consisting of deoxyribonucleic acids (DNA), ribonucleic acids (RNA), or synthetic nucleic acids such as. As the peptide nucleic acids (PNA) and the Locked nucleic acids (LNA) exist.

When Oligonucleotide primer (or primer for short) is usually an oligonucleotide with about 12 to 30 nucleotides, which is complementary to a Section of a larger DNA or RNA molecule is and about a free 3'OH group at its 3'-end features. Due to this free OH group, the primer can be used as a substrate for DNA polymerases in a polymerase chain reaction (PCR), the DNA polymerase in 5'-3 'direction nucleotides synthesized to the primer. The sequence of the newly synthesized Nucleotide is thereby hybridized by the sequence of the primer Given target DNA, which is beyond the free 3'OH group of the primer. The im Primers used in this invention preferably include 12 and 50 nucleotides, more preferably between 15 and 30 nucleotides and most preferably between 18 and 28 nucleotides.

A Sequence complementary to a nucleotide sequence provides a sequence on nucleotides in a single strand, forming specific Base pairings hybridized with a single strand, which the having said nucleotide sequence.

Under Deletion is understood as a loss of a nucleotide sequence section wherein the ends of the adjacent areas connect with each other. Under addition will be an additional Nucleotide sequence to which a Vergleichnukleotidsequenz was extended. Substitution is an exchange of one or more nucleotides in a nucleotide sequence.

Under a target nucleic acid, Target nucleic acid sequence, Target DNA or shortly a target is within the scope of the present Invention in particular a present in the genome of Salmonella enterica nucleic acid sequence understood the evidence of the identity of a present in the sample Serovars of the species Salmonella enterica suspec. enterica supplies and amplified in the PCR. In accordance with the invention, it is target nucleic acid sequences to sequences present in the genes STM3356, sopE1 or avrA.

When PCR product becomes a product within the scope of the present invention from the amplification of the nucleic acid molecules to be amplified the PCR designates.

When Amplification phase is in the context of the present invention, the Phase referred to, in which the PCR takes place.

Under the denaturation temperature is within the scope of the present invention understood the temperature at which the double-stranded DNA separated in the PCR cycle becomes. The denaturation temperature is usually more than 90 ° C, preferably about 95 ° C. Below the annealing temperature is used in the context of the present invention the temperature at which the primers hybridize to the target. The annealing temperature is usually in the range of 30 ° C up to 65 ° C and is preferably about 45 ° C. Under the chain extension or extension temperature is within the scope of the present invention understood the temperature at which the DNA by incorporation of the monomer building blocks is synthesized. The extension temperature is usually in the range of about 70 ° C up to about 75 ° C and is preferably about 72 ° C.

As used herein refers to an oligonucleotide probe (or, in short: Probe) an oligonucleotide used for the detection of a specific Nucleotide sequence is used in a target DNA. The detection is done by specific hybridization of the probe with a single strand the target DNA. The probe has at least over a sequence region that is complementary to a sequence region of the target nucleic acid. The The probes used in the invention preferably have one length of 15 to 70 nucleotides, more preferably from 18 to 60 nucleotides and most preferably from 18 to 55 nucleotides. The can Oligonukleotidsonden also on a solid support substrate in the form of a biochip be immobilized.

Under Hybridization is in the context of the present invention, the association two single-stranded nucleic acid fragments understood to a double strand. The association preferably always finds to pairs of A and T and G and C, respectively.

According to the invention is a Hybridization in vitro always be carried out under conditions which are stringent enough to allow for specific hybridization guarantee. Such stringent hybridization conditions are those skilled in the art known and can from the literature (Sambrook et al., 2001, Molecular Cloning: A laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press).

Generally means "specific hybridize", that a molecule under stringent conditions preferential binds to a particular nucleotide sequence when that sequence is in a complex mixture of (e.g., total) DNA or RNA. The term "stringent Conditions " generally for Conditions under which a nucleic acid sequence is preferential will hybridize to their target sequence, and to a much smaller one Extent or at all not to other sequences. Stringent conditions are z.T. Sequence-dependent and be under different circumstances be different. longer Sequences hybridize specifically at higher temperatures.

In general, stringent conditions are selected such that the temperature is about 5 ° C below the thermal melting point (T _m ) for the specific sequence at a defined ionic strength and pH. The T _m is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the molecules complementary to the target sequence hybridize to the target sequence in the equilibrium state. Typically, stringent conditions are those in which the salt concentration is at least about 0.01 to 1.0 M sodium ion concentration (or another salt) at a pH between 7.0 and 8.3 and the temperature is at least about 30 ° C short molecules (eg 10-50 nucleotides). In addition, stringent conditions, as mentioned above, can be achieved by adding destabilizing agents such as formamide.

Typical hybridization and washing buffers have, for example, the following composition: prehybridization solution: 0.5% SDS 5 × SSC 50 mM NaPO ₄ , pH 6.8 0.1% Na pyrophosphate 5 × Denhardt's solution 100 μg / ml salmon sperm Hybridization solution: Prähybridisierungslsg. 1 × 10 ⁶ cpm / ml probe (5-10 min 95 ° C) 20 × SSC: 3M NaCl 0.3 M sodium citrate ad pH 7 with HCl 50 × Denhardt's reagent: 5 g Ficoll 5 g of polyvinylpyrrolidone 5 g Bovine Serum Albumine ad 500 ml A. dist.

A typical procedure for hybridization is as follows:
Optional: Wash blot for 30 min in 1 x SSC / 0.1% SDS at 65 ° C
Prehybridization: at least 2 h at 50-55 ° C
Hybridization: overnight at 55-60 ° C

Of the Professional knows that the solutions mentioned and the protocol shown may be modified depending on the application can or must.

Alternatively, the person skilled in the art can also determine by means of a conventional PCR whether the primer oligonucleotides he has chosen specifically bind to a specific target sequence by generating the PCR product checking whether a distinct fragment of the expected length is formed.

Under Real-time PCR or real-time PCR is generally understood as the Analysis of amplification of a target DNA in a PCR procedure by detecting fluorescence from a dye, either directly or indirectly with the amplification of specifically amplified DNA is associated.

at A multiplex PCR simultaneously becomes several specific primer pairs used and with their help in a vessel just as many different Target nucleic acids amplified.

According to the invention is a Procedure provided for the detection of the two serovars Java and Paratyphi B of the species Salmonella enterica subspec. enterica suitable. In addition, oligonucleotide probes, oligonucleotide primers and kits are provided for use in the method of the invention. The oligonucleotide probes according to the invention and oligonucleotide primers, which are particularly suitable for use in a real-time multiplex PCR for selective Propagation of target DNA and for subsequent detection, are each of nucleotide sequences of the chromosomal genes STM3356, sopE1 and avrA, derived. Furthermore, the invention comprises a Biochip on which the probes according to the invention in immobilized form available.

One significant advantage of the invention over the prior art in the comparatively short time necessary for the reliable distinction between the two serovars is needed and less than one Working day. Other advantages are the possibility the use of the primer according to the invention and probes in modern real-time PCR technology, simplification of the verification process and the increase in cost efficiency the preferred combination of 4 detection systems in a single, so-called 4-way multiplex reaction and especially in the combination the detection of different, with the pathogenicity of the pathogens correlating factors to increase of specificity and reliability of Proof.

The polymerase chain reaction (PCR) is a common and well-known molecular biology method for amplification of fewer moles of any genomic DNA sequence in a very short time in vitro by factors of 10 ⁶ to 10 ⁸ . The real-time PCR derived therefrom allows analysis of the amplification by detection of fluorescence from a dye associated either directly or indirectly with the amplification of the specifically amplified DNA (see review in Journal of Molecular Endocrinology 2000 vol 25, pp. 169-193).

For the implementation of Real-time PCR offers various techniques. The original version of the Real-time PCR uses a dye that nonspecifically intercalates into duplexes and then fluoresces. In the propagation of the target DNA binds the dye attaches to the newly formed DNA duplexes, which leads to a measurable increase in fluorescence.

Of the Use of fluorescence resonance energy transfer (FRET) probes, which also on double-stranded DNA binding is another direct method. A FRET probe is a short nucleotide sequence that is complementary to one of the target genome sequence strands. The probe is covalent with a so-called reporter at the 5'-end and with a so-called quencher at the 3'-end connected. In the unbound state of the probe become the dyes through a complementary Loop arrangement in spatial Kept close. complementary Sequences at the ends of the probe sequence thereby generate the loop arrangement. The quencher dye is capable of being close to the reporter to quench its fluorescence by the FRET. The probe is also called "molecular called beacon " and is combined with the corresponding primer pair in a real-time PCR used. By binding the probe to the amplified target DNA the loop arrangement is canceled and the two dyes become spatially separated, eliminating the interference and the fluorescence the reporter dye is measurable.

Holland et al. describe a so-called Tagman procedure (Holland, P.M., Abramson, R.D., Watson, R., and Gelfand, D.H. 1991. Detection of specific polymerase chain reaction product by utilizing the 5'-3 'exonuclease activity of Thermus aguaticus DNA polymerase. Proc. Natl. Acad. Sci. 88: 7276-7280). However, the FRET probe used here has no Loop arrangement. While the polymerase replicates the strand of DNA, the exo-nuclease activity builds the FRET probe, which is bound to the target DNA, at its 5'-end and the reporter dye is released from the probe. As a result, the reporter dye is no longer spatial near of the quencher dye, so that its fluorescence is no longer extinguished and is now measurable. Propagation of the target DNA and the associated release of the reporter dye is detectable by suitable measuring systems.

One Another indirect method consists of the combination of the mentioned Beacons with the primers, with a primer via a non-amplifiable Connect to the beacon via its 5'-end connected is. In the amplification of the target DNA by the PCR is this Probe, also called Scorpion, connected to the target DNA sequence, but not self-amplified because of the non-amplifiable compound. In the following denaturation step, that to the target DNA complementary Probe by breaking the loop order with the target DNA subsequent cooling tie. With the departure, the spatial proximity of the two dyes is prevented and the fluorescence of the reporter dye becomes measurable. For further Explanations is published on Science, Vol 296, pages 557-58, 19 April 2002 and Journal of Molecular Endocrinology 2002, 29, pp. 23-29 and www.dxsgenotyping.com directed.

• – Amplifizieren eines Bereichs des Salmonella-Gens STM3356 unter Verwendung eines Oligonukleotidprimerpaares bestehend aus einem ersten und einem zweiten Primer, die in der Lage sind, mit Sequenzen des Salmonella-Gens STM3356 spezifisch zu hybridisieren, und
• – Hybridisieren einer ersten Oligonukleotidsonde, umfassend mindestens 18, bevorzugt mindestens 20, 22, 25, 28 und am meisten bevorzugt mindestens 30, 35, 40, 45 fortlaufende Nukleotide aus der Nukleotidsequenz SEQ ID Nr. 1 oder der dazu komplementären Sequenz oder mindestens 18, bevorzugt mindestens 20, 22, 25, 28 und am meisten bevorzugt mindestens 30, 35, 40, 45 Nukleotide aus einer Nukleotidsequenz, die sich von der Nukleotidsequenz SEQ ID Nr. 1 oder der dazu komplementären Sequenz durch eine Deletion, Addition und/oder Substitution von bis zu 3, bevorzugt 2, am meisten bevorzugt 1 Nukleotid unterscheidet, wobei diese erste Sonde in der Lage ist, mit einem Bereich des Salmonella-Gens STM3356 spezifisch zu hybridisieren, und mindestens die Nukleotidabfolge gemäß SEQ ID Nr. 2 oder der dazu komplementären Sequenz aufweist, mit dem amplifizierten Bereich des Salmonella-Gens, und/oder
• – Hybridisieren einer zweiten Oligonukleotidsonde, umfassend mindestens 18, bevorzugt mindestens 20, 22, 25, 28 und am meisten bevorzugt mindestens 30, 35, 40, 45 fortlaufende Nukleotide aus der Nukleotidsequenz SEQ ID Nr. 3 oder der dazu komplementären Sequenz oder mindestens 18, bevorzugt mindestens 20, 22, 25, 28 und am meisten bevorzugt mindestens 30, 35, 40, 45 Nukleotide aus einer Nukleotidsequenz, die sich von der Nukleotidsequenz SEQ ID Nr. 3 oder der dazu komplementären Sequenz durch eine Deletion, Addition und/oder Substitution von bis zu 3, bevorzugt 2 und besonders bevorzugt 1 Nukleotid unterscheidet, wobei diese zweite Sonde in der Lage ist, mit einem Bereich des Salmonella-Gens STM3356 spezifisch zu hybridisieren, und mindestens die Nukleotidabfolge gemäß SEQ ID Nr. 4 oder der dazu komplementären Sequenz aufweist, mit dem amplifizierten Bereich des Salmonella-Gens,

wobei der amplifizierte Bereich des Salmonella-Gens STM3356 mindestens die SEQ ID Nr. 2 oder 4 aufweist.According to the invention, there is provided a method for detecting and / or distinguishing Salmonella serovars, the method comprising:

• Amplifying a region of the Salmonella gene STM3356 using an oligonucleotide primer pair consisting of a first and a second primer capable of specifically hybridizing with sequences of the Salmonella gene STM3356, and
• Hybridizing a first oligonucleotide probe comprising at least 18, preferably at least 20, 22, 25, 28 and most preferably at least 30, 35, 40, 45 contiguous nucleotides from the nucleotide sequence SEQ ID No. 1 or the complementary sequence or at least 18, preferably at least 20, 22, 25, 28 and most preferably at least 30, 35, 40, 45 nucleotides from a nucleotide sequence different from the nucleotide sequence SEQ ID NO: 1 or the sequence complementary thereto by deletion, addition and / or substitution of up to 3, preferably 2, most preferably 1 nucleotide, wherein said first probe is capable of specifically hybridizing with a region of the Salmonella gene STM3356, and at least the nucleotide sequence of SEQ ID NO: 2 or the complementary thereto Sequence, with the amplified region of the Salmonella gene, and / or
• Hybridizing a second oligonucleotide probe comprising at least 18, preferably at least 20, 22, 25, 28 and most preferably at least 30, 35, 40, 45 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 3 or the complementary sequence or at least 18, preferably at least 20, 22, 25, 28 and most preferably at least 30, 35, 40, 45 nucleotides from a nucleotide sequence different from the nucleotide sequence SEQ ID NO: 3 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3, preferably 2 and more preferably 1 nucleotide, this second probe being able to specifically hybridize with a region of the Salmonella gene STM3356, and at least the nucleotide sequence according to SEQ ID NO: 4 or the sequence complementary thereto having, with the amplified region of the Salmonella gene,

wherein the amplified region of the Salmonella gene STM3356 has at least the SEQ ID NO: 2 or 4.

If the gene STM3356 of Salmonella enterica amplified as a target gene should be, so can different Primers are used as long as they contain a single amplicon So generate PCR product containing the sequence to be detected contains. The expert is the problem of the optimal primer design, that is, the search for the best nucleotide sequence for primer synthesis at a given target DNA sequence known. The primers decide generally over Success and failure of the real-time PCR according to the invention, because with incorrectly constructed primers can fail the process because either at all no synthesis takes place or because a wrong fragment or even multiple DNA sections of the sample DNA are amplified. The oligonucleotide primers recommended here are specifically in this described method, in particular in the multiplex method especially to be preferred.

• – einem ersten Oligonukleotidprimer, der mindestens 15, bevorzugt mindestens 17, 19 und besonders bevorzugt mindestens 20, 21 fortlaufende Nukleotide aus der Nukleotidsequenz SEQ ID Nr. 5 oder der dazu komplementären Sequenz oder mindestens 15, bevorzugt mindestens 17, 19 und besonders bevorzugt mindestens 20, 21 Nukleotide aus einer Nukleotidsequenz umfasst, die sich von der Nukleotidsequenz SEQ ID Nr. 5 oder der dazu komplementären Sequenz durch eine Deletion, Addition und/oder Substitution von bis zu 3, bevorzugt 2 und am meisten bevorzugt 1 Nukleotid unterscheidet, und
• – einem zweiten Oligonukleotidprimer, der mindestens 15, bevorzugt mindestens 17, 19 und besonders bevorzugt mindestens 20, 21 fortlaufende Nukleotide aus der Nukleotidsequenz SEQ ID Nr. 6 oder der dazu komplementären Sequenz oder mindestens 15, bevorzugt mindestens 17, 19 und besonders bevorzugt mindestens 20, 21 Nukleotide aus einer Nukleotidsequenz umfasst, die sich von der Nukleotidsequenz SEQ ID Nr. 6 oder der dazu komplementären Sequenz durch eine Deletion, Addition und/oder Substitution von bis zu 3, bevorzugt 2 und am meisten bevorzugt 1 Nukleotid unterscheidet.

The oligonucleotide primer pair consists of a preferred embodiment

• A first oligonucleotide primer which has at least 15, preferably at least 17, 19 and particularly preferably at least 20, 21 consecutive nucleotides from the nucleotide sequence SEQ ID NO. 5 or the complementary sequence or at least 15, preferably at least 17, 19 and particularly preferably at least 20 21 comprises nucleotides from a nucleotide sequence different from the nucleotide sequence SEQ ID NO: 5 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3, preferably 2 and most preferably 1 nucleotide, and
• A second oligonucleotide primer which has at least 15, preferably at least 17, 19 and particularly preferably at least 20, 21 contiguous nucleotides from the nucleotide sequence SEQ ID No. 6 or the complementary sequence or at least 15, preferably at least 17, 19 and particularly preferably at least 20 , 21 nucleotides from a nucleotide sequence which differs from the nucleotide sequence SEQ ID No. 6 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3, preferably 2 and most preferably 1 nucleotide.

The Oligonucleotide primers flank a nucleotide sequence region the gene STM3356. In a real-time PCR method is thus a Part of the gene STM3356 amplified, which either the ATG start codon or a mutation in this codon (ATA). The variance of the polymorphic Locus correlates with the presence Salmonella Java or Salmonella Paratyhi B in the examined and inserted sample.

to Identification of the nucleotide sequence of typhic gene STM3356 Serovars Paratyhpi B uses the first oligonucleotide probe. Furthermore, for the simultaneous identification of the nucleic acid sequence of the enteric serovar Java gene the second oligonucleotide probe used.

at The oligonucleotide probes of the invention may be DNA or RNA probes that are between 10 and 100 nucleotides of the gene STM3356 and more preferably between 15 and 70 nucleotides. Similarly, as a probe, the corresponding complementary sequence used equally well with a single strand of the DNA segment of gene STM3356 hybridized. The nucleotide sequence the probe does not necessarily have to be completely complementary to the target DNA and can also be by deletion, addition and / or substitution of up to 3 nucleotides from those given in the Sequence Listing Distinguish sequences. The properties mentioned apply as well for probes according to the invention, which are described below.

Preferably, the probes are covalently linked to a so-called reporter at the 5 'end and to a so-called quencher at the 3' end. As a detectable reporter dye, also called a marker, fluorescent groups are used such. FAM (6-carboxyfluorescein, available from Sigma Aldrich, Deisenhofen), HEX (hexachloro-6-carboxyfluorescein, available from Molecular Probes, Leiden), Texas Red (sulforhodamine sulfonyl chloride, available from Molecular Probes, Leiden), Cy5 (NHS ester Cy5.18 (sulfoindocyanine succinimidyl ester), available from Molecular Probes, Leiden), Cy3 (NHS ester from Cy5.18, available from Molecular Probes, Leiden), Cy2 (NHS ester from Cy5.18, available from Molecular Probes, Leiden ), FITC (fluorescein isothiocyanate, available from Sigma Aldrich, Deisenhofen), CT (5, (6) -carboxytetramethylrhodamine N-hydroxysuccinimide ester, available from Molecular Probes, Leiden), TRITC (tetramethylrhodamine-5,6-isothiocyanate, available from Sigma Aldrich , Deisenhofen), 5-ROX (5-carboxy-X-rhodamine, available from Molecular Probes, Leiden), ITC (isothiocyanate, available from Molecular Probes, Leiden), Fluos-Prime (5,6-carboxyfluorescein-N-hydroxysuccinimide ester, available from Sigma Aldrich, Deisenhofen), 6-TET (6-carboxy-2 ', 4,7,7'-tetrachlorofluorescein, available from Molecular Probes, Leiden) or other fluorescent groups not listed herein that can be "quenched" via interference and known to those skilled in the art. Some markers, their properties and possible sources of supply are given in the following Table 1: TABLE I

In a preferred embodiment is the first oligonucleotide probe with the compound FAM and the second oligonucleotide probe connected to the compound HEX.

When Quenchers are dyes in question such. Dabcyl (4- (4-dimethylaminophenylazo) benzoate, available from Molecular Probes, Leiden), 6-TAMRA (6-carboxytetramethylrhodamine, available from Molecular Probes, Leiden), Black Hole Quenchers (4 '- (2-Toluyldiazo) -2'-methylazobenzene-4' '- (N-ethyl) -N-ethyl-2-cyanoethyl- (N, N-diisopropyl) phosphoramidite, available by Jena Bioscience, Jena) or other quencher known to those skilled in the art, which can quench one of the above markers by interference.

In a preferred embodiment are both the first oligonucleotide probe and the second oligonucleotide probe with the quencher Dabcyl at the 3'-end connected.

in the unbound state of the probes, the dyes are replaced by a complementary Loop arrangement in spatial Kept close. The complementary ones Sequences at the ends of the probe sequence thereby generate the loop arrangement. The probes in the present invention have the sequence at the 5 'end CGCGATC, which uses the loop by internal hybridization Watson-and-Crick Bassenpaarungen with the sequence GATCGCG at the 3 'end forms. Of the Quencher dye is capable of being close to the reporter its fluorescence extinguish.

Prefers The first oligonucleotide probe and the second probe are combined used with the primer pair described above in a real-time PCR.

• – die Denaturierungstemperatur beträgt 95 °C für einen Zeitraum von 14 Minuten 30 Sekunden,
• – es folgen 50 PCR-Zyklen mit jeweils einer Denaturierungstemperatur von 95 °C für 30 Sekunden, einer Annealingtemperatur von 45 °C für eine Minute und einer Kettenverlängerungstemperatur von 72 °C für 30 Sekunden.

The phases of a real-time PCR process are generally known to the person skilled in the art. The amplification phase is carried out according to the present invention in a preferred embodiment under the following conditions:

• The denaturation temperature is 95 ° C for a period of 14 minutes 30 seconds,
• Followed by 50 PCR cycles, each with a denaturation temperature of 95 ° C for 30 seconds, an annealing temperature of 45 ° C for one minute and a chain extension temperature of 72 ° C for 30 seconds.

At close the amplification phase a denaturation step (95 ° C for one minute) and a renaturation step on (from 80 ° C after 30 ° C in increments of one degree per minute). The two strands of the amplified DNA separates rapidly when the hydrogen bonds between the base pairs are broken. This is done by heating to 95 ° C. The separate complementary strands encase themselves with the complementary ones Probes back together when the temperature is lowered below the melting temperature becomes. The melting curve to be detected is determined by the fluorescence intensity of the probes taken into account in the evaluation of the results.

By Annealing the probe to the amplified target DNA the previously described loop arrangement canceled and the two Dyes become spatial separated, eliminating the interference and the fluorescence the reporter dye is measurable. In the preferred concurrent use Care should be taken to ensure that the fluorescent reporters, the be used to label the probes, in their emission spectrum can be distinguished photometrically. Detection is via fluorescence measurements, d. H. the product formation is due to the fluorescence increases of the Dyes (e.g., FAM and HEX) photometrically using a photometer, that in the PCR device integrated, measured. For this purpose, the basis of the Amplifikationsverläufe Threshold set at which the fluorescent signal of the reporter dye clearly over the background signal and the amplification of the target DNA under non-limiting conditions (linear range). In an embodiment the fluorescence signals of PCR cycles 5-12 of the amplification phase measured and evaluated as unspecific background signal. The ten times the standard deviation is added to the mean and as a threshold for determine the determination of the Ct value. The Ct value thus corresponds to the number of PCR cycles, at the fluorescence intensity reached the threshold. For Evidence of a mutation in the start codon of STM3356 gene is included the evaluation of both the Ct value of the fluorescent dyes, as also the curve thus the increase of the fluorescence intensity and the Melting curve of importance.

• – Der Ct-Wert des Markers der ersten Oligonukleotidsonde (hier FAM) ist kleiner als der Ct-Wert des Markers der zweiten Oligonukleotidsonde (hier HEX).
• – Anstieg der Kurve im mittleren Teil bei FAM > HEX, d. h. die Fluoreszenzintensität von FAM nimmt mit der Entstehung in Form einer Kurve zu; der Anstieg der Fluoreszenzintensität im mittleren Teil der Kurve ist bei FAM größer als der Anstieg der Fluoreszenzintensität von HEX (siehe 1).
• – Die Schmelzkurve von FAM fällt zwischen dem 40. und dem 50. Zyklus stärker ab (bzw. nimmt weniger stark zu) als bei HEX. Hierzu wird der Abschnitt zwischen 40 °C und 30 °C betrachtet (siehe 2).

When assaying a sample using the method of the invention, I am Salmonella Paratyphi B when at least two out of three conditions are met:

• The Ct value of the marker of the first oligonucleotide probe (here FAM) is smaller than the Ct value of the marker of the second oligonucleotide probe (here HEX).
• - increase of the curve in the middle part in FAM> HEX, ie the fluorescence intensity of FAM increases with the formation in the form of a curve; the increase in fluorescence intensity in the middle part of the curve is greater in FAM than the increase in fluorescence intensity of HEX (see 1 ).
• - The melting curve of FAM falls more sharply between the 40th and the 50th cycle (or increases less) than in HEX. For this purpose, the section between 40 ° C and 30 ° C is considered (see 2 ).

• – Der Ct-Wert des Markers der zweiten Oligonukleotidsonde (hier HEX) ist kleiner als der Ct-Wert des Markers der ersten Oligonukleotidsonde (hier FAM).
• – Anstieg der Kurve im mittleren Teil bei HEX > FAM, d. h. die Fluoreszenzintensität von HEX nimmt mit der Entstehung in Form einer Kurve zu; der Anstieg der Fluoreszenzintensität im mittleren Teil der Kurve ist bei HEX größer als der Anstieg der Fluoreszenzintensität von FAM (siehe 3).
• – Die Schmelzkurve von HEX fällt zwischen dem 40. und dem 50. Zyklus stärker ab (bzw. nimmt weniger stark zu) als bei FAM. Hierzu wird der Abschnitt zwischen 40 °C und 30 °C betrachtet (siehe 4).

When testing a sample using the method of the invention, I am dealing with Salmonella Java when at least two out of three conditions are met:

• The Ct value of the marker of the second oligonucleotide probe (here HEX) is smaller than the Ct value of the marker of the first oligonucleotide probe (here FAM).
• - increase of the curve in the middle part in HEX> FAM, ie the fluorescence intensity of HEX increases with the formation in the form of a curve; the increase in fluorescence intensity in the middle part of the curve is greater in HEX than the increase in fluorescence intensity of FAM (see 3 ).
• - The melting curve of HEX falls more sharply between the 40th and the 50th cycle (or increases less strongly) than in FAM. For this purpose, the section between 40 ° C and 30 ° C is considered (see 4 ).

In the present method for distinguishing the two serovars Java and Paratyphi B of the species Salmonella enterica subspec. enterica amplification of the polymorphic gene STM 3356 is first performed with DNA sequence-specific detection in a single PCR tube. For the first time a fast procedure is described, with which the discrimination and identification of the serovars Java and Paratyphi B is possible in one and the same PCR tube. Lengthy cultivation methods, as required in the prior art, are not necessary because of the low detection limit of the method according to the invention, nor have the amplificates as bands in a subsequent gel electropho rese be proven. The associated time requirement and also the risk of contamination already mentioned can thus be ruled out for the first time. Advantages of this method over prior art methods are also the very easy handling, since in this method all steps are carried out in one and the same PCR vessel, and on the other the economy, since here detection systems are brought together in a single multiplex process ,

The serve in the present invention provided primer pairs the amplification of the above-mentioned target gene region in one PCR method. In particular, the primer pairs are in their length each other adapted to facilitate PCR in a multiplexed approach, i. H. with the method according to the invention and the primer nucleotide sequences employed therein as well as those described herein Probes it is possible a sample in a "pot" on the or Absence of several genes to investigate. According to the invention Examination of the genotypic variance of the gene STM3356 with the Detection of the genes sopE1 and avrA combined for the first time.

• – Amplifizieren eines Bereichs des Salmonella-Gens sopE1 unter Verwendung eines Oligonukleotidprimerpaares bestehend aus einem dritten und einem vierten Primer, die in der Lage sind, mit Sequenzen des Salmonella-Gens sopE1 spezifisch zu hybridisieren, und
• – Amplifizieren eines Bereichs des Salmonella-Gens avrA unter Verwendung eines Oligonukleotidprimerpaares bestehend aus einem fünften und einem sechsten Primer, die in der Lage sind, mit Sequenzen des Salmonella-Gens avrA spezifisch zu hybridisieren, und
• – Hybridisieren einer dritten Oligonukleotidsonde, umfassend mindestens 18, bevorzugt mindestens 20, 22 und am meisten bevorzugt 24, 26, 28 fortlaufende Nukleotide aus der Nukleotidsequenz SEQ ID Nr. 7 oder der dazu komplementären Sequenz oder mindestens 18, bevorzugt mindestens 20, 22 und am meisten bevorzugt 24, 26, 28 Nukleotide aus einer Nukleotidsequenz, die sich von der Nukleotidsequenz SEQ ID Nr. 7 oder der dazu komplementären Sequenz durch eine Deletion, Addition und/oder Substitution von bis zu 3, bevorzugt 2 und am meisten bevorzugt 1 Nukleotid unterscheidet, wobei die dritte Sonde in der Lage ist, mit einem Bereich des Salmonella-Gens sopE1 spezifisch zu hybridisieren, mit dem amplifizierten Bereich des Salmonella-Gens sopE1, und
• – Hybridisieren einer vierten Oligonukleotidsonde, umfassend mindestens 18, bevorzugt mindestens 22, 24, 26, und am meisten bevorzugt 30, 34, 38 fortlaufende Nukleotide aus der Nukleotidsequenz SEQ ID Nr. 8 oder der dazu komplementären Sequenz oder mindestens 18, bevorzugt mindestens 22, 24, 26, und am meisten bevorzugt 30, 34, 38 Nukleotide aus einer Nukleotidsequenz, die sich von der Nukleotidsequenz SEQ ID Nr. 8 oder der dazu komplementären Sequenz durch eine Deletion, Addition und/oder Substitution von bis zu 3, bevorzugt 2 und am meisten bevorzugt 1 Nukleotid unterscheidet, wobei die vierte Sonde in der Lage ist, mit einem Bereich des Salmonella- Gens avrA spezifisch zu hybridisieren, mit dem amplifizierten Bereich des Salmonella-Gens avrA.

A further embodiment of the present invention therefore further comprises

• Amplifying a region of the Salmonella gene sopE1 using an oligonucleotide primer pair consisting of a third and a fourth primer which are capable of specifically hybridizing to sequences of the Salmonella gene sopE1, and
• Amplifying a region of the Salmonella gene avrA using an oligonucleotide primer pair consisting of a fifth and a sixth primer which are capable of specifically hybridizing with sequences of the Salmonella gene avrA, and
• Hybridizing a third oligonucleotide probe comprising at least 18, preferably at least 20, 22 and most preferably 24, 26, 28 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 7 or its complementary sequence or at least 18, preferably at least 20, 22 and most preferably 24, 26, 28 nucleotides from a nucleotide sequence different from the nucleotide sequence SEQ ID NO: 7 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3, preferably 2 and most preferably 1 nucleotide wherein the third probe is capable of specifically hybridizing with a region of the Salmonella gene sopE1, with the amplified region of the Salmonella gene sopE1, and
• Hybridizing a fourth oligonucleotide probe comprising at least 18, preferably at least 22, 24, 26, and most preferably 30, 34, 38 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 8 or its complementary sequence or at least 18, preferably at least 22, 24, 26, and most preferably 30, 34, 38 nucleotides from a nucleotide sequence different from the nucleotide sequence SEQ ID NO: 8 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3, preferably 2 and most preferably 1 nucleotide, wherein the fourth probe is capable of specifically hybridizing with a region of the Salmonella gene avrA, with the amplified region of the Salmonella gene avrA.

at the most preferred simultaneous use of the first, second, third and fourth probes in a multiplexed approach ensure that fluorescent reporters are used to label the corresponding probes are used in their emission spectra can be distinguished photometrically. The third oligonucleotide probe and the fourth oligonucleotide probe are preferably those for the first and second oligonucleotide probe referred to properties on the characteristic loop arrangement. In one embodiment is the third oligonucleotide probe at the 5 'end with the marker Texas Red and at the 3' end with the compound Dabcyl as a quencher dye and the fourth oligonucleotide probe at the 5'-end with the Compound Cy5 as reporter dye and at the 3 'end connected to the Black Hole Quencher.

• – einem dritten Oligonukleotidprimer, der mindestens 15, bevorzugt 17, 19 und besonders bevorzugt mindestens 20, 21 fortlaufende Nukleotide aus der Nukleotidsequenz SEQ ID Nr. 9 oder der dazu komplementären Sequenz oder mindestens 15, bevorzugt mindestens 17, 19 und besonders bevorzugt mindestens 20, 21 Nukleotide aus einer Nukleotidsequenz umfasst, die sich von der Nukleotidsequenz SEQ ID Nr. 9 oder der dazu komplementären Sequenz durch eine Deletion, Addition und/oder Substitution von bis zu 3, bevorzugt 2 und meisten bevorzugt 1 Nukleotid unterscheidet, und
• – einem vierten Oligonukleotidprimer, der mindestens 15, bevorzugt 17, 19 und besonders bevorzugt 20, 21 fortlaufende Nukleotide aus der Nukleotidsequenz SEQ ID Nr. 10 oder der dazu komplementären Sequenz oder mindestens 15, bevorzugt 17, 19 und besonders bevorzugt 20, 21 Nukleotide aus einer Nukleotidsequenz umfasst, die sich von der

For the amplification of the target gene region of sopE1 of Salmonella enterica, in a particularly preferred embodiment, the following oligonucleotide primer pair is used, consisting of

• A third oligonucleotide primer which has at least 15, preferably 17, 19 and particularly preferably at least 20, 21 consecutive nucleotides from the nucleotide sequence SEQ ID No. 9 or the complementary sequence or at least 15, preferably at least 17, 19 and particularly preferably at least 20, 21 nucleotides from a nucleotide sequence which differs from the nucleotide sequence SEQ ID No. 9 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3, preferably 2 and most preferably 1 nucleotide, and
• - A fourth oligonucleotide primer, the at least 15, preferably 17, 19 and particularly preferably 20, 21 contiguous nucleotides from the nucleotide sequence SEQ ID NO. 10 or the complementary sequence or at least 15, preferably 17, 19 and particularly preferably 20, 21 nucleotides a nucleotide sequence other than the

nucleotide SEQ ID NO: 10 or the sequence complementary thereto by a deletion, Addition and / or substitution of up to 3, preferably 2 and on most preferably 1 nucleotide is different.

• – einem fünften Oligonukleotidprimer, der mindestens 15, bevorzugt mindestens 17, 18 und besonders bevorzugt mindestens 19, 20 fortlaufende Nukleotide aus der Nukleotidsequenz SEQ ID Nr. 11 oder der dazu komplementären Sequenz oder mindestens 15, bevorzugt 17, 18 und besonders bevorzugt mindestens 19, 20 Nukleotide aus einer Nukleotidsequenz umfasst, die sich von der Nukleotidsequenz SEQ ID Nr. 11 oder der dazu komplementären Sequenz durch eine Deletion, Addition und/oder Substitution von bis zu 3, bevorzugt 2 und am meisten bevorzugt 1 Nukleotid unterscheidet, und
• – einem sechsten Oligonukleotidprimer, der mindestens 15, bevorzugt 17, 20 und besonders bevorzugt 22, 24 fortlaufende Nukleotide aus der Nukleotidsequenz SEQ ID Nr. 12 oder der dazu komplementären Sequenz oder mindestens 15, bevorzugt 17, 20 und besonders bevorzugt 22, 24 Nukleotide aus einer Nukleotidsequenz umfasst, die sich von der Nukleotidsequenz SEQ ID Nr. 12 oder der dazu komplementären Sequenz durch eine Deletion, Addition und/oder Substitution von bis zu 3, bevorzugt 2 und am meisten bevorzugt 1 Nukleotid unterscheidet.

For the amplification of the target gene region of avrA of Salmonella enterica, in a particularly preferred embodiment, an oligonucleotide primer pair consisting of

• A fifth oligonucleotide primer which has at least 15, preferably at least 17, 18 and particularly preferably at least 19, 20 consecutive nucleotides from the nucleotide sequence SEQ ID No. 11 or the sequence complementary thereto or at least 15, preferably 17, 18 and particularly preferably at least 19, 20 nucleotides from a nucleotide sequence which differs from the nucleotide sequence SEQ ID NO: 11 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3, preferably 2 and most preferably 1 nucleotide, and
• - A sixth oligonucleotide primer, the at least 15, preferably 17, 20 and particularly preferably 22, 24 contiguous nucleotides from the nucleotide sequence SEQ ID NO. 12 or the complementary sequence or at least 15, preferably 17, 20 and particularly preferably 22, 24 nucleotides a nucleotide sequence which differs from the nucleotide sequence SEQ ID NO: 12 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3, preferably 2 and most preferably 1 nucleotide.

• – nur Texas Red (im Ausführungsbeispiel die dritte Sonde) (siehe 5).

The typhoid serovars have the sopE1-containing bacteriophage, but not the avrA gene in their genome, whereas enteric serovars are heterogeneous in this respect: they may either have only the avrA gene or the avrA gene in combination with the sopE1 gene or none of them two genes. Accordingly, the measured fluorescence values of the third and fourth oligonucleotide probes are evaluated as follows:
It is Salmonella Paratyphi B if the following probes show a Ct value and a sigmoid curve:

• - Only Texas Red (in the embodiment, the third probe) (see 5 ).

• – nur Cy5 (im Ausführungsbeispiel die vierte Sonde) (siehe 6)
• – Texas Red und Cy5 (im Ausführungsbeispiel die dritte und vierte Sonde)
• – keine von beiden.

It is Salmonella Java if the following probes show a Ct value and a sigmoid curve:

• - Only Cy5 (in the embodiment, the fourth probe) (see 6 )
• Texas Red and Cy5 (third and fourth probe in the embodiment)
• - neither.

According to the invention simultaneous use of an oligonucleotide primer pair consisting of a first primer according to SEQ ID No. 5 and a second primer according to SEQ ID No. 6, one Oligonucleotide primer pair consisting of a third primer according to SEQ ID No. 9 and a fourth primer according to SEQ ID No. 10 and an oligonucleotide primer pair, that from a fifth Primer according to SEQ ID No. 11 and a sixth primer according to SEQ ID NO: 12 examining a sample using the present method most preferred. In this case, the above-described amplification phase in a preferred embodiment can not be customized because the primers have already been chosen that they are in their length and kinetics are matched.

The Ct values of the probes are determined by standard. The genesis of the products is measured photometrically as described above.

Of the Detection of genes sopE1 and avrA with the help of the third and fourth Oligonucleotide probe provides another, of the first and second Oligonucleotide probe independent This combination of proof of two independent, with pathogenicity the pathogen correlating factors does not allow one until then achieved specificity and speed of proof.

The inventive method can be varied be applied. A field of application for the present method is the control of food samples. In preferred embodiments the samples come from milk or dairy products (yoghurt, cottage cheese, Cheese, butter, Buttermilk), drinking water, drinks (juices, lemonade, Beer), ice cream, baked goods or meat products, eggs or other Foods that may contain salmonella. For the detection of Salmonella in food can possibly a previous enrichment desired or even prescribed. So it is necessary z. B. for the proof of single salmonella in 25 ml of milk to cultivate this for a while, afterwards also with statistical reliability to have one or more Salmonella in the sample volume.

The method according to the invention can further be used for the examination of medical samples become. It is useful for examining tissue samples, blood and other body fluids, as well as stool and urine samples. For the detection of Salmonella from clinical material may be desired or even prescribed prior enrichment or cultivation of the bacteria under certain circumstances.

One Another important application for the present method is the study of wastewater. About that It is also suitable for analyzing biofilms in industrial plants. as well as naturally forming biofilms or biofilms forming wastewater treatment to investigate. Finally is It also used for examination and quality control of pharmaceutical and cosmetic products, e.g. As ointments, creams, tinctures, juices etc. suitable.

The inventive probe systems are chosen that they are based on substrates can be immobilized. These are usually gold, glass, silicon compounds, etc., which are known to those skilled in the art as carriers for microarrays are known. Very particular are the probe systems of the invention for the Use on biochips. So the probes z. B. via nanospotters on carrier applied and immobilized. The inventive method can thus be advantageously using biochips be automated. Thus, the probes of the invention with the corresponding Primers for Several genes could be used simultaneously on biochips safe according to the invention provided that their kinetic properties approximate each other, i. H. that they are multiplexable are. Otherwise could falsified results for a particular gene can be obtained if the associated primer pair z. B. a cheaper Kinetics than the rest.

According to the invention is at a further aspect of the present invention, a kit for carrying out the Method for detecting and / or distinguishing the above Serovars of Salmonella enterica are provided in a sample. Such a kit comprises as the most important ingredient at least one first probe for the gene STM3356 from Salmonella enterica subsp. enterica serovar Parathyphi B and / or a second probe for the same Salmonella gene enterica subsp. enterica Serovar Java and prefers a first and a second probe to use either one as a control. In a further embodiment contains the Kit about it In addition, a first oligonucleotide primer according to SEQ ID NO. 5 and a second Oligonucleotide primer according to SEQ ID No. 6 for the specific amplification of a nucleotide sequence section of the gene STM3356. In a preferred embodiment, the kit contains in addition to the first and second STM3356-specific oligonucleotide probe and the first and second primers one specific for the gene sopE1 (third) oligonucleotide probe, as well as one specific for the gene avrA (fourth) oligonucleotide probe. The most preferred embodiment of the kit includes all oligonucleotide probes listed above and all listed above Oligonucleotide primer for the application of the invention in a multiplex real-time PCR for the selective amplification of target DNA of the genes STM3356, sopE1 and avrA and to the subsequent specific Detection.

Farther will carry a kit of the method for the detection of the above mentioned serovars of Salmonella enterica provided in a sample containing one for the gene sopE1-specific oligonucleotide probe and one for the gene avrA specific oligonucleotide probe. In a preferred embodiment In this kit, these oligonucleotide probes are used together with the oligonucleotide primers according to the sequences SEQ ID Nos. 9-12.

The The following example and illustrations are for explanation of the invention and are not to be construed in a limiting manner:

Example: identification and differentiation of Salmonella enterica subspec. enterica serovar Paratyphi B and Salmonella enterica subspec. enterica Serovar Java in a sample

Preparation of the samples

A Salmonella enterica subspec. Enterica Serovar Paratyphi B sample and Salmonella enterica subspec. Enterica Serovar Java containing sample was used for DNA isolation. DNA extraction was performed using the commercially available QIAamp DNA Mini Kit (from QIAgen, Hilden) and included the following steps:
The sample suspension was incubated at 95 ° C for 20 min.

The Bacterial suspension was purified by centrifugation at 10,000 x g (ca. 10 000-11 000 rpm) for 15 minutes. pelleted in a standard tabletop centrifuge.

The supernatant was discarded, the bacterial pellet was in 180 ul ATL buffer resuspended, 20 μl proteinase K were added, vortexed briefly and incubated at 70 ° C for 10 min.

The Sample was briefly spun down, 200 μl of AL buffer was added and for about. 15 s vortext.

The Sample was spun down briefly, 200 μl of ethanol (96-100%) became admit and for about 15 s vortext.

One Filter tube was placed in a collecting vessel and the solution out pipetted to the previous step in the upper reservoir.

The Filter tube was sealed and left for 1 min at 6000 xg (ca. 8000 rpm).

Of the Pass was discarded, the filter tube was placed back into the collecting vessel, 500 μl AW1 buffer were pipetted and then again for 1 min. centrifuged at 6000 x g.

Of the Pass was discarded, the filter tube put back into the collecting vessel, 500 μl AW2 buffer pipetted and for 3 min at highest Centrifuged speed.

The Collecting vessel was discarded and the filter tube inserted into a clean 1.5 ml reaction vessel.

to Elution of the DNA was 400 μl AE buffer pipetted into the filter tube, incubated for 1 min at room temperature and subsequently 1 min at 6000 x g centrifuged. The filter tube was discarded; the DNA was in the eluate and later as a DNA solution in the method according to the invention used.

The DNA can also be used with other methods known to those skilled in the art DNA isolation or with other commercially available kits.

Primer / probe design

The sequences of the genes were taken from the NCBI database (GenBank):
The sequence of STM3356 is originally from the strain Salmonella enterica Serovar Typhimurium LT2. From this sequence, the sequence of Serovar Paratyhpi B differs by the above nucleotide exchange in the start codon.

All Primers and fluorogenic probes were from commercial suppliers based. The specificity the primer and probes were analyzed by sequence comparison using the BLAST method with the NCBI entries determined.

PCR

• – 5 μl DNA-Lösung, wobei die DNA in Puffer AE (QIAamp DNA Mini Kit von Qiagen) resuspendiert vorlag,
• – 5 μl 10 × PCR-Puffer, enthaltend 15 mM MgCl₂ (von Qiagen, Hilden),
• – 11 μl einer 25 mM MgCl₂ (von Qiagen, Hilden),
• – 2 μl einer 50 μM Primer-Lösung, wobei der Primer die Nukleotidsequenz gemäß SEQ ID Nr. 5 aufwies,
• – 2 μl einer 50 μM Primer-Lösung, wobei der Primer die Nukleotidsequenz gemäß SEQ ID Nr. 6 aufwies,
• – 2 μl einer 50 μM Primer-Lösung, wobei der Primer die Nukleotidsequenz gemäß SEQ ID Nr. 9 aufwies,
• – 2 μl einer 50 μM Primer-Lösung, wobei der Primer die Nukleotidsequenz gemäß SEQ ID Nr. 10 aufwies,
• – 2 μl einer 50 μM Primer-Lösung, wobei der Primer die Nukleotidsequenz gemäß SEQ ID Nr. 11 aufwies,
• – 2 μl einer 50 μM Primer-Lösung, wobei der Primer die Nukleotidsequenz gemäß SEQ ID Nr. 12 aufwies,
• – 0,1 μl einer 50 μM Sonden-Lösung, wobei die Sonde die Nukleotidsequenz gemäß SEQ ID Nr. 1 und am 5'-Ende den Marker FAM und am 3'-Ende den Quencher Dabcyl aufwies,
• – 0,4 μl einer 50 μM Sonden-Lösung, wobei die Sonde die Nukleotidsequenz gemäß SEQ ID Nr. 3 und am 5'-Ende den Marker HEX und am 3'-Ende den Quencher Dabcyl aufwies,
• – 0,2 μl einer 50 μM Sonden-Lösung, wobei die Sonde die Nukleotidsequenz gemäß SEQ ID Nr. 7 und am 5'-Ende den Marker Texas Red und am 3'-Ende den Quencher Dabcyl aufwies,
• – 0,2 μl einer 50 μM Sonden-Lösung, wobei die Sonde die Nukleotidsequenz gemäß SEQ ID Nr. 8 und am 5'-Ende den Marker Cy5 und am 3'-Ende den Black Hole Quencher aufwies,
• – 0,3 μl einer 25 mM dNTP-Lösung (Roth, Karlsruhe),
• – 0,5 μl HotstarTaq DNA-Polymerase (mit einer Volumenaktivität von 5 U/μl, Qiagen, Hilden) und
• – 15,3 μl destilliertes Wasser.

A PCR batch in a PCR tube contained the following components:

• 5 μl of DNA solution, whereby the DNA was resuspended in buffer AE (QIAamp DNA Mini Kit from Qiagen),
• 5 μl of 10 × PCR buffer containing 15 mM MgCl ₂ (ex Qiagen, Hilden),
• 11 μl of a 25 mM MgCl ₂ (from Qiagen, Hilden),
• 2 μl of a 50 μM primer solution, the primer having the nucleotide sequence according to SEQ ID No. 5,
• 2 μl of a 50 μM primer solution, the primer having the nucleotide sequence according to SEQ ID No. 6,
• 2 μl of a 50 μM primer solution, the primer having the nucleotide sequence according to SEQ ID No. 9,
• 2 μl of a 50 μM primer solution, the primer having the nucleotide sequence according to SEQ ID No. 10,
• 2 μl of a 50 μM primer solution, the primer having the nucleotide sequence according to SEQ ID No. 11,
• 2 μl of a 50 μM primer solution, the primer having the nucleotide sequence according to SEQ ID No. 12,
• 0.1 μl of a 50 μM probe solution, the probe having the nucleotide sequence according to SEQ ID No. 1 and the marker FAM at the 5 'end and the quencher dabcyl at the 3' end,
• 0.4 μl of a 50 μM probe solution, the probe having the nucleotide sequence according to SEQ ID No. 3 and the marker HEX at the 5 'end and the quencher dabcyl at the 3' end,
• 0.2 μl of a 50 μM probe solution, the probe having the nucleotide sequence according to SEQ ID No. 7 and the marker Texas Red at the 5 'end and the quencher dabcyl at the 3' end,
• 0.2 μl of a 50 μM probe solution, the probe having the nucleotide sequence according to SEQ ID No. 8 and the marker Cy5 at the 5 'end and the black hole quencher at the 3' end,
• 0.3 μl of a 25 mM dNTP solution (Roth, Karlsruhe),
• - 0.5 μl of HotstarTaq DNA polymerase (with a volume activity of 5 U / μl, Qiagen, Hilden) and
• - 15.3 μl of distilled water.

This PCR approach was performed in Microcups (ICN Biomedical, Ohio) in a Mx3000P PCR thermocycler (Stratagene, Amsterdam) are subjected to the following temperature profile: 14 min 30 sec at 95 ° C, then 50 cycles with each 30 sec at 95 ° C, 1 min at 45 ° C and 30 sec at 72 ° C, then 1 min at 95 ° C. it The restoration followed from 80 ° C to 30 ° C in increments of each Degrees per minute.

detection and identification

The used oligonucleotide probes were determined by their fluorescence reporter, which were used to mark the corresponding probes, identified photometrically. PCR product formation was during the PCR course by the fluorescence increases of the dyes FAM (at a wavelength about 517 nm), HEX (at a wavelength of about 556 nm), Texas Red (at one wavelength of about 615 nm) and Cy5 (at a wavelength of about 670 nm) photometrically followed in the above-mentioned PCR thermocycler and recorded the data. Based on the Amplifikationsverläufe a threshold was set at which the respective fluorescence signal the reporter dye was well above the background signal and amplification of the target DNA under non-linear conditions expired (exponential range). The fluorescence signals of the PCR cycles 5-12 of the amplification phase were measured and scored as a background signal. The tenfold standard deviation was added to the mean and as a threshold for determine the determination of the respective Ct value.

• – Der Ct-Wert für FAM hatte einen Wert von 17,5 und war somit kleiner als der Ct-Wert für HEX (19,2) (siehe die angegebenen Ct-Werte in 7).
• – Der Anstieg der Fluoreszenzintensität von FAM war im mittleren Teil der Kurve größer als der Anstieg der Fluoreszenzintensität von HEX. Dies fiel auf, wenn man die Anstiege zwischen dem 20. und dem 40. Zyklus der Kurven betrachtete (siehe 7).
• – Die Schmelzkurve von FAM fiel im Temperaturbereich zwischen 40 °C und 30 °C stärker ab, verglichen mit der Schmelzkurve von HEX (siehe 2).
• – Nur Texas Red (TEX) wies einen Ct-Wert (17,2) und eine sigmoide Kurve auf, Cy5 hingegen nicht (siehe 7).

For the detection or differentiation of the Salmonella serovars Paratyphi B and Java, both the Ct values of the fluorescent dyes as well as the curves of the fluorescence intensities and the melting curves were analyzed. Salmonella enterica subspec. enterica serovar paratyphi B, which was contained in one of the two samples, was identified as such by the method according to the invention, since the following detection conditions were fulfilled:

• The Ct value for FAM was 17.5, which was smaller than the Ct value for HEX (19.2) (see the Ct values given in FIG 7 ).
• The increase in fluorescence intensity of FAM in the middle part of the curve was greater than the increase in the fluorescence intensity of HEX. This was noticeable when looking at the climbs between the 20th and the 40th cycle of the turns (see 7 ).
• - The melting curve of FAM decreased more in the temperature range between 40 ° C and 30 ° C compared to the melting curve of HEX (see 2 ).
• - Only Texas Red (TEX) had a Ct value (17.2) and a sigmoid curve, while Cy5 did not (see 7 ).

In summary it became clear that the detection method independent features, namely the initial described mutation in the start codon of the gene STM3356 and the presence of the gene sopE1 and the known serotype Paratyphi B can identify as such.

• – Der ermittelte Ct-Wert für HEX betrug 18,4. Für FAM konnte jedoch kein Ct-Wert ermittelt werden, so dass kein größerer von einem kleineren Ct-Wert unterschieden wurde (siehe 8). Die zuvor genannte Bedingung, dass der Ct-Wert von HEX kleiner als der Ct-Wert von FAM sein muss, wurde somit nicht erfüllt, doch müssen auch nur zwei von drei Bedingungen für den Nachweis des Genotyps von Gen STM3356 erfüllt sein.
• – Der Anstieg der Fluoreszenzintensität von HEX war im mittleren Teil der Kurve größer als der Anstieg der Fluoreszenzintensität von FAM (siehe 8).
• – Die Schmelzkurve von HEX fiel im Temperaturbereich zwischen 40 °C und 30 °C stärker ab, verglichen mit der Schmelzkurve von FAM (siehe 4).
• – Nur Cy5 wies einen Ct-Wert (16,6) und eine sigmoide Kurve auf, TEX hingegen nicht (siehe 8).

Salmonella enterica subspec. enterica serovar Java, which was included in the further sample, was also identified by the method according to the invention:

• The calculated Ct value for HEX was 18.4. For FAM, however, no Ct value could be determined so that no larger one was distinguished from a smaller Ct value (see 8th ). The aforementioned condition that the Ct value of HEX must be smaller than the Ct value of FAM was thus not met, but only two out of three conditions for the detection of the genotype of STM3356 gene must be met.
• The increase in the fluorescence intensity of HEX was greater than the increase in the mean part of the curve Fluorescence intensity of FAM (see 8th ).
• - The melting curve of HEX fell more sharply in the temperature range between 40 ° C and 30 ° C compared to the melting curve of FAM (see 4 ).
• - Only Cy5 had a Ct value (16.6) and a sigmoid curve, whereas TEX did not (see 8th ).

Also Here it became clear that the detection method according to the invention the genotype of the gene STM3356 and the presence of the gene avrA detect and can identify the known serotype Java as such.

In order to test the reliability of the method according to the invention, a validation of the method described here was carried out on 50 D-tartrate-positive and 50 D-tartrate-negative isolates. The results of the validation are shown in Table 2 below. TABLE 2

Based The results clearly show that the STM3356-related detection Completely and the sopE1 / avrA-related detection correlates 98% with the D-tartrate phenotype. From 100 isolates only 2 isolates showed discrepancies regarding a unique assignment. These discrepant cases can with Help of the method according to the invention by the simultaneous consideration The described factors are recognized, which in turn is alone Consideration of the sopE1 / avrA result would not be possible.

pictures:

1 2 shows the increase of fluorescence intensities for the fluorescent dye FAM of the first oligonucleotide probe (data points shown in the form of diamonds) and for the fluorescent dye HEX of the second oligonucleotide probe (data points in the form of triangles) in a positive detection of Salmonella Paratyphi B. The abscissa axis denotes the measured Fluorescence intensity and the ordinate axis indicate the number of cycles. Other unmarked curves shown here are not to be considered in this context.

2 shows the melting curve over the fluorescence intensity for the fluorescent dye FAM of the first oligonucleotide probe (data points shown in the form of diamonds) and for the fluorescent dye HEX of the second oligonucleotide probe (data points shown in the form of triangles) in a positive detection of Salmonella Paratyphi B. The abscissa axis denotes the measured fluorescence intensity and the ordinate axis indicates the temperature (° C). The temperature range between 40 ° C and 30 ° C is indicated by the two arrows on the ordinate axis. Other unmarked curves shown here are not to be considered in this context.

3 shows the increase in fluorescence intensities for the fluorescent dye FAM of the first oligonucleotide probe (data points shown in the form of diamonds) and for the fluorescent dye HEX of the second oligonucleotide probe (data points shown in the form of triangles) in a positive detection of Salmonella Java. The abscissa axis indicates the measured fluorescence intensity and the ordinate axis indicates the number of cycles. Other unmarked curves shown here are not to be considered in this context.

4 shows the melting curve over the fluorescence intensity for the fluorescent dye FAM of the first oligonucleotide probe (data points shown in the form of diamonds) and for the fluorescent dye HEX of the second oligonucleotide probe (data points shown in the form of triangles) in a positive detection of Salmonella Java. The abscissa axis indicates the measured fluorescence intensity and the ordinate axis indicates the temperature (° C). The temperature range between 40 ° C and 30 ° C is indicated by the two arrows on the ordinate axis. Other shown here, not marked Ned curves are not to be considered in this context.

5 2 shows the increase of the fluorescence intensities for the fluorescence dye Texas Red (TEX) of the third oligonucleotide probe (data points shown in the form of squares) and for the fluorescence dye Cy5 of the fourth oligonucleotide probe (data points in the form of circles) in a positive detection of Salmonella paratyphi B. Die The axis of abscissa denotes the measured fluorescence intensity and the ordinate axis indicates the number of cycles. Other unmarked curves shown here are not to be considered in this context.

6 shows the increase in fluorescence intensities for the fluorescent dye Cy5 of the fourth oligonucleotide probe (data points shown in the form of circles) in a positive detection of Salmonella Java. An increase in the fluorescence intensity of the fluorescent dye Texas Red (TEX) of the third oligonucleotide probe (data points shown in the form of squares) did not occur in this case. The abscissa axis indicates the measured fluorescence intensity and the ordinate axis indicates the number of cycles. Other unmarked curves shown here are not to be considered in this context.

7 Figure 4 shows the increase in fluorescence intensities for the fluorescent dye FAM of the first oligonucleotide probe (data points shown in the form of diamonds) for the fluorescent dye HEX of the second oligonucleotide probe (data points shown in the form of triangles) for the fluorescent dye Texas Red (TEX) of the third oligonucleotide probe (data points in the form of squares) and no increase for the fluorescence dye Cy5 of the fourth oligonucleotide probe (data points shown in the form of circles) in a positive detection of Salmonella Paratyphi B. The abscissa axis denotes the measured fluorescence intensity and the ordinate axis indicates the number of cycles. The corresponding Ct values of said fluorescent dyes are indicated.

8th 1 shows the increase of the fluorescence intensities for the fluorescence dye FAM of the first oligonucleotide probe (data points shown in the form of diamonds), for the fluorescence dye HEX of the second oligonucleotide probe (data points shown in the form of triangles) and for the fluorescence dye Cy5 of the fourth oligonucleotide probe (data points in the form of circles shown) with a positive detection of Salmonella Java. An increase in the fluorescence intensity of the fluorescent dye Texas Red (TEX) of the third oligonucleotide probe (data points shown in the form of squares) did not occur in this case. The abscissa axis indicates the measured fluorescence intensity and the ordinate axis indicates the number of cycles. The corresponding Ct values of said fluorescent dyes are indicated. sequences

Claims (18)

Method for detection and / or differentiation of Salmonella serovars comprising: - Amplify a range Salmonella STM3356 using an oligonucleotide primer pair consisting of a first and a second primer in the Capable of being specific with sequences of the Salmonella gene STM3356 to hybridize, and - Hybridize a first oligonucleotide probe comprising at least 18 consecutive Nucleotides from the nucleotide sequence SEQ ID NO: 1 or the complementary nucleotide sequence or at least 18 nucleotides from a sequence different from the Nucleotide sequence SEQ ID NO: 1 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3 Nucleotides, this first probe being able to specifically to hybridize with a region of the Salmonella gene STM3356, and at least the nucleotide sequence according to SEQ ID NO. 2 or the thereto complementary Sequence, with the amplified region of the Salmonella gene and or - Hybridize a second oligonucleotide probe comprising at least 18 consecutive Nucleotides from the nucleotide sequence SEQ ID NO. 3 or the sequence complementary thereto or at least 18 nucleotides from a nucleotide sequence that are from the nucleotide sequence SEQ ID NO: 3 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3 Nucleotides differs, with this second probe being able to is specific to a region of the Salmonella gene STM3356 hybridize, and at least the nucleotide sequence according to SEQ ID No. 4 or the complementary thereto Sequence with the amplified region of the Salmonella gene, in which the amplified region of the Salmonella gene STM3356 at least SEQ ID NO: 2 or 4. The method of claim 1, wherein: - the first Oligonucleotide primers at least 15 contiguous nucleotides from the Nucleotide sequence SEQ ID NO: 5 or its complementary sequence or at least 15 nucleotides from a nucleotide sequence, that is different from the nucleotide sequence SEQ ID NO. 5 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3 Nucleotides differs, and The second oligonucleotide primer at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID No. 6 or its complementary sequence or at least Comprises 15 nucleotides from a nucleotide sequence different from the Nucleotide sequence SEQ ID NO. 6 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides is different. Method according to one of claims 1 and 2, further comprising: - Amplify a portion of the Salmonella gene sopE1 using a Oligonucleotide primer pair consisting of a third and a fourth primers that are capable of sequences of the Salmonella gene specifically to hybridize sopE1, and - Amplify a range of the Salmonella gene avrA using an oligonucleotide primer pair consisting of a fifth and a sixth primer capable of containing sequences of the Salmonella gene avrA specifically to hybridize, and - Hybridize a third oligonucleotide probe comprising at least 18 consecutive Nucleotides from the nucleotide sequence SEQ ID No. 7 or the sequence complementary thereto or at least 18 nucleotides from a nucleotide sequence that are from the nucleotide sequence SEQ ID NO: 7 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides, the third probe being able to specifically to hybridize with a region of the Salmonella gene sopE1, with the amplified region of the Salmonella gene sopE1 and - Hybridize a fourth oligonucleotide probe comprising at least 18 consecutive Nucleotides from the nucleotide sequence SEQ ID No. 8 or the sequence complementary thereto or at least 18 nucleotides from a nucleotide sequence that are from the nucleotide sequence SEQ ID NO: 8 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides, with the fourth probe being able to specifically to hybridize with a region of the Salmonella gene avrA, with the amplified region of the Salmonella gene avrA. The method of claim 3, wherein - the third Oligonucleotide primers at least 15 contiguous nucleotides from the Nucleotide sequence SEQ ID NO. 9 or its complementary sequence or at least 15 nucleotides from a nucleotide sequence, which differ from the nucleotide sequence SEQ ID No. 9 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3 Nucleotides differs, and The fourth oligonucleotide primer at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID No. 10 or its complementary sequence or at least Comprises 15 nucleotides from a nucleotide sequence different from the Nucleotide sequence SEQ ID NO: 10 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides differs, and - the fifth oligonucleotide primer at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 11 or the complementary one Sequence or at least 15 nucleotides from a nucleotide sequence which is different from the nucleotide sequence SEQ ID NO. 11 or the complementary to it Sequence by a deletion, addition and / or substitution of differentiates up to 3 nucleotides, and The sixth oligonucleotide primer at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID NO. 12 or its complementary sequence or at least Comprises 15 nucleotides from a nucleotide sequence different from the Nucleotide sequence SEQ ID NO: 12 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides is different. The method of claim 3 or 4, wherein the first Oligonucleotide primer, the second oligonucleotide primer, the third Oligonucleotide primer, the fourth oligonucleotide primer, the fifth oligonucleotide primer and the sixth oligonucleotide primer together with the first oligonucleotide probe, the second oligonucleotide probe, the third oligonucleotide probe and the fourth oligonucleotide probe used in a real-time multiplex PCR become. A method of detecting and / or distinguishing Salmonella serovars comprising: - amplifying a region of the Salmonella gene sopE1 using a primer pair of oligonucleotide primers capable of being specific with sequences of the Salmonella gene sopE1 hybridizing, and - amplifying a region of the Salmonella gene avrA using an oligonucleotide primer pair consisting of a third and a fourth primer capable of specifically hybridizing with sequences of the Salmonella gene avrA, and hybridizing a first oligonucleotide probe, comprising at least 18 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 7 or its complementary sequence, or at least 18 nucleotides from a nucleotide sequence, different from the nucleotide sequence SEQ ID NO: 7 or the sequence complementary thereto by deletion, addition and / or substitution of up to 3 nucleotides below This first probe is capable of specifically hybridizing with a region of the Salmonella gene sopE1, with the amplified region of the Salmonella gene sopE1 and - hybridizing a second oligonucleotide probe comprising at least 18 contiguous nucleotides from the nucleotide sequence SEQ ID NO 8 or the sequence complementary thereto or at least 18 nucleotides from a nucleotide sequence which differ from the nucleotide sequence SEQ ID No. 8 or the complementary Se quence by a deletion, addition and / or substitution of up to 3 nucleotides, said second probe being able to specifically hybridize with a region of the Salmonella gene avrA, with the amplified region of the Salmonella gene avrA. The method of claim 6, wherein: - the first Oligonucleotide primers at least 15 contiguous nucleotides from the Nucleotide sequence SEQ ID NO. 9 or its complementary sequence or at least 15 nucleotides from a nucleotide sequence, which differ from the nucleotide sequence SEQ ID No. 9 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3 Nucleotides differs, and The second oligonucleotide primer at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID No. 10 or its complementary sequence or at least Comprises 15 nucleotides from a nucleotide sequence different from the Nucleotide sequence SEQ ID NO: 10 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides differs, and The third oligonucleotide primer at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID NO. 11 or its complementary sequence or at least Comprises 15 nucleotides from a nucleotide sequence different from the Nucleotide sequence SEQ ID NO: 11 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides differs, and The fourth oligonucleotide primer at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID NO. 12 or its complementary sequence or at least Comprises 15 nucleotides from a nucleotide sequence different from the Nucleotide sequence SEQ ID NO: 12 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides is different. Method according to one of the preceding claims, wherein the Salmonella serovars "Paratyphi B "and" Java "of the subspecies Salmonella enterica subspec. enterica are distinguished from each other. Method according to one of the preceding claims, wherein the oligonucleotide probes are immobilized on a support. Biochip for the detection and / or differentiation of Salmonella serovars, the biochip comprising a carrier with at least one on the carrier immobilized oligonucleotide probe, wherein the immobilized oligonucleotide probe selected is made up of the group An oligonucleotide probe, comprising at least 18 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 1 or the complementary sequence or at least 18 nucleotides from a nucleotide sequence different from the nucleotide sequence SEQ ID NO: 1 or the sequence complementary thereto by a deletion, Addition and / or substitution of up to 3 nucleotides distinguishes, this probe being capable of interfering with a region of the Salmonella gene STM3356 specifically to hybridize, and at least the nucleotide sequence according to SEQ ID No. 2 or the complementary thereto Sequence, and - one An oligonucleotide probe comprising at least 18 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 3 or the sequence complementary thereto or at least 18 nucleotides from a nucleotide sequence that are from the nucleotide sequence SEQ ID NO: 3 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3 Nucleotides, this probe being able to with specifically to hybridize to a region of the Salmonella gene STM3356, and at least the nucleotide sequence according to SEQ ID NO. 4 or the thereto complementary Sequence has. The biochip of claim 10, wherein the carrier further includes as immobilized probes An oligonucleotide probe, comprising at least 18 contiguous nucleotides from the nucleotide sequence SEQ ID No. 7 or its complementary sequence or at least 18 nucleotides from a nucleotide sequence different from the nucleotide sequence SEQ ID NO: 7 or its complementary sequence by a deletion, Addition and / or substitution of up to 3 nucleotides distinguishes, this probe being capable of interfering with a region of the Salmonella gene specifically to hybridize sopE1, and An oligonucleotide probe, comprising at least 18 contiguous nucleotides from the nucleotide sequence SEQ ID No. 8 or its complementary sequence or at least 18 nucleotides from a nucleotide sequence different from the nucleotide sequence SEQ ID NO: 8 or the sequence complementary thereto by a deletion, Addition and / or substitution of up to 3 nucleotides distinguishes, this probe being capable of interfering with a region of the Salmonella gene avrA specifically to hybridize. An oligonucleotide which specifically hybridizes to a region of a Salmonella gene selected from STM3356, sopE1 and avrA and at least 15 contiguous nucleotides from a nucleotide sequence according to SEQ ID Nos. 1, 3, 5, 6, 7, 8, 9, 10, 11 and 12 or a sequence complementary thereto or at least 15 nucleotides comprising a nucleotide sequence which is different from a nucleotide sequence according to SEQ ID No. 1, 3, 5, 6, 7, 8, 9, 10, 11 and 12 or a sequence complementary thereto by a deletion, addition and / or substitution of up to 3 nucleotides. Kit for the detection and / or differentiation of Salmonella serovars comprising: A first oligonucleotide probe, comprising at least 18 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 1 or its complementary sequence or at least 18 nucleotides from a nucleotide sequence different from the nucleotide sequence SEQ ID NO: 1 or the sequence complementary thereto by a deletion, Addition and / or substitution of up to 3 nucleotides distinguishes, wherein said first probe is capable of interfering with a region of the Salmonella gene STM3356 specifically to hybridize, and at least the nucleotide sequence according to SEQ ID No. 2 or the complementary thereto Sequence and / or - one second oligonucleotide probe comprising at least 18 consecutive Nucleotides from the nucleotide sequence SEQ ID NO. 3 or the sequence complementary thereto or at least 18 nucleotides from a nucleotide sequence that are from the nucleotide sequence SEQ ID NO: 3 or the sequence complementary thereto by a deletion, addition and / or substitution of up to 3 Nucleotides differs, with this second probe being able to is specific to a region of the Salmonella gene STM3356 hybridize, and at least the nucleotide sequence according to SEQ ID No. 4 or the complementary thereto Sequence has. The kit of claim 13, further comprising: - one first oligonucleotide primer comprising at least 15 consecutive Nucleotides from the nucleotide sequence SEQ ID NO. 5 or the sequence complementary thereto or at least 15 nucleotides from a sequence different from the Nucleotide sequence SEQ ID NO: 5 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides differs, and A second oligonucleotide primer, comprising at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 6 or its complementary sequence or at least 15 nucleotides from a sequence different from the nucleotide sequence SEQ ID NO: 6 or the sequence complementary thereto by a deletion, Addition and / or substitution of up to 3 nucleotides differs. A kit according to claim 13 or 14, further comprising: - a third An oligonucleotide probe comprising at least 18 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 7 or the sequence complementary thereto or at least 18 nucleotides from a sequence different from the Nucleotide sequence SEQ ID NO: 7 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides, the third probe being able to specifically to hybridize with a region of the Salmonella gene sopE1, and - one fourth oligonucleotide probe comprising at least 18 consecutive Nucleotides from the nucleotide sequence SEQ ID No. 8 or the sequence complementary thereto or at least 18 nucleotides from a sequence different from the Nucleotide sequence SEQ ID NO: 8 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides, with the fourth probe being able to specifically to hybridize with a region of the Salmonella gene avrA. The kit of claim 15, further comprising: - one third oligonucleotide primer comprising at least 15 consecutive Nucleotides from the nucleotide sequence SEQ ID No. 9 or the sequence complementary thereto or at least 15 nucleotides from a sequence different from the Nucleotide sequence SEQ ID NO. 9 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides differs, and A fourth oligonucleotide primer, comprising at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 10 or its complementary sequence or at least 15 nucleotides from a sequence different from the nucleotide sequence SEQ ID NO: 10 or the sequence complementary thereto by a deletion, Addition and / or substitution of up to 3 nucleotides distinguishes, and - one fifth An oligonucleotide primer comprising at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 11 or the sequence complementary thereto or at least 15 nucleotides from a sequence different from the Nucleotide sequence SEQ ID NO: 11 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides differs, and A sixth oligonucleotide primer, comprising at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 12 or its complementary sequence or at least 15 nucleotides from a sequence different from the nucleotide sequence SEQ ID NO: 12 or the sequence complementary thereto by a deletion, Addition and / or substitution of up to 3 nucleotides differs. A kit for the detection and differentiation of Salmonella serovars, comprising: an oligonucleotide probe comprising at least 18 contiguous nucleotides from the nucleotide sequence SEQ ID No. 7 or the sequence complementary thereto or at least 18 nucleotides from a sequence, the differs from the nucleotide sequence SEQ ID NO: 7 or its complementary sequence by a deletion, addition and / or substitution of up to 3 nucleotides, which probe is capable of specifically hybridizing with a region of the Salmonella gene sopE1, and - an oligonucleotide probe comprising at least 18 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 8 or its complementary sequence or at least 18 nucleotides from a sequence other than the nucleotide sequence SEQ ID NO: 8 or the sequence complementary thereto by a deletion, Addition and / or substitution of up to 3 nucleotides, this probe being able to specifically hybridize with a region of the Salmonella gene avrA. The kit of claim 17, further comprising: - one An oligonucleotide primer comprising at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID NO. 9 or the sequence complementary thereto or at least 15 nucleotides from a sequence different from the Nucleotide sequence SEQ ID NO. 9 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides differs, and An oligonucleotide primer, comprising at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID No. 10 or its complementary sequence or at least 15 nucleotides from a sequence different from the nucleotide sequence SEQ ID NO: 10 or the sequence complementary thereto by a deletion, Addition and / or substitution of up to 3 nucleotides distinguishes, and - one An oligonucleotide primer comprising at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID NO: 11 or the sequence complementary thereto or at least 15 nucleotides from a sequence different from the Nucleotide sequence SEQ ID NO: 11 or its complementary sequence by a deletion, addition and / or substitution of up to 3 Nucleotides differs, and An oligonucleotide primer, comprising at least 15 contiguous nucleotides from the nucleotide sequence SEQ ID NO. 12 or its complementary sequence or at least 15 nucleotides from a sequence different from the nucleotide sequence SEQ ID NO: 12 or the sequence complementary thereto by a deletion, Addition and / or substitution of up to 3 nucleotides differs.