GB2500757A - Analytical method and kits for detecting nucleic acids on non-biological surfaces - Google Patents

Abstract

A method for detecting the presence of a nucleic acid in the presence of environmental contaminants on a non-biological surface, said method comprising applying to anabsorbent body that has been in contact with the non-biological surface, a volume of an amplification reaction mixture that is at least sufficient to saturate the absorbent body in a reaction vessel; carrying out an amplification reaction in said vessel, and detecting amplified nucleic acid produced. The absorbent body is small, for example a neonatal swab, so as to fit in a standard 200ul PCR reaction tube and the method may be used to detect trace DNA samples, which may be analysed for example in DNA profiling. The non biological surface may be an object such as a mobile phone. Kits for use in the method are also described and claimed.

Description

Analytical Method The present invention relates to a method for detecting a nucleic acid in the presence of environmental contaminants, in particular in trace samples such as those that are used in DNA analysis such as forensic analysis or hygiene or other monitoring, as well as to kits and reagents for use in the method.

Analysis of nucleic acids is now a widely used technique in a variety of fields including diagnostics, genotyping, hygiene monitoring, environmental monitoring and detection and forensics.

Human DNA forensics is a worldwide multi-billion dollar industry. The most technically challenging work in DNA forensics is to recover trace human DNA samples left at crime scenes or on objects, and amplify this trace DNA to create a DNA profile. Conventional techniques require trace DNA to be swabbed from an object, purified and concentrated, quantified and finally amplified and analysed. The DNA purification, concentration and quantification steps for trace DNA require specialist equipment, lengthy protocols, ultra-dean reagents and specialist staff Trace DNA forensic analysis is usually carried out by using a wetted swab to swab an object thought to be contaminated with a suspect's DNA. Any fl-ace DNA on the object is absorbed onto the swab, together with any contaminants from the environment, such as humic acids or metal ion chelators that are present on the surface.

It is recognised that these contaminants will prevent amplification of the DNA using conventional techniques such as the polymerase chain reaction IPCR), and so the DNA must be extracted and purified before further analysis.

This is generally done using specialist DNA purification laboratory kits and equipment (e.g. Qiagen EZI or Appfied Biosystems® PrepFiler'). The manual steps involved can take from 30-60 minutes to execute. The resulting trace DNA extract is usually very dilute and is required to be concentrated before it can be used in an amplification reaction such as PCR.

Each of these operations increases the risk of further contamination of the sample with DNA for example from the operator or the environment. Furthermore, the purification process is inefficient, and a proportion of the trace DNA may be lost.

Since the amount present in the first place is extremely low, further losses may cause the amplification reaction to fail.

I

Recent developments in the field of direct DNA amplification methods have focused on the specific reagents used in the methods to reduce the need for sample preparation. For instance, US2010/0015621 describes methods for conducting PCR on "crude" reference samples such as Hood samples or buccal cdl samples taken from a biological subject, using a specific buffer. The samples are either stored on FTA® (Whatman®) paper (which in fact contain a cocktail of reagents that lyse cells, denature proteins and preserve nucleic acids) or diluted with or extracted into TE buffer prior to analysis using the direct buffer. Thus although reducing the intial process steps, this technology does not eliminate preparation steps altogether, and produces a dilute sample of DNA.

Kits for carrying out such analysis on reference samples such as criminal database samp'es, paternity samples and casework reference samples using this technology are available from Applied Biosystems® under the tradename Identifiler® Direct. In this case, biological samples (such as blood or buccal samples) are spotted onto ETA paper and the subsequent amplification and analysis is carried out using a sample punched from the paper.

It has been recognized in the art that this methodology is not applicable to trace DNA samples that are found in DNA forensics (see review in van Oorschot et al., Tnvestigative Genetics 2010, 1:14 page 5 column 2). For those samples. work has been focused on trying to obtain a more efficient extraction and purification procedure from media such as the swab, used to collect the trace DNA sample.

Though a recent report by Swaran et al., Forensic Science International: Genetics, 6.2012.407-412, has purportivdy compared the result of direct PCR with PCR following extraction this has only been undertaken under artificial conditions, wherein punfied nucleic acid has been deposited on cleaned surfaces (section 2.

Materials and Methods), and thus critically not in the presence of environmental contaminants, and thus is not concerned with the widely recognised problem of inhibition of nucleic acid amplification reactions by environmental contaminants.

Trace DNA analysis is therefore difficult to carry out quickly, especially so in

the field.

The applicants have found a method by which DNA samples and particularly trace DNA samples can be analyzed efficiently and with minimum risk of contamination.

According to the present invention there is provided a method for detecting the presence of a nucleic acid in the presence of environmental contaminants on a non-biological surface, said method comprising applying to an absorbent body that has been in contact with the non-biological surface, a volume of an amplification reaction mixture that is at least sufficient to saturate the absorbent body in a reaction vessel; carrying out an amplification reaction in said vessel, and detecting amplified nucleic acid produced.

The appficants have found that a nucleic acid amplification reaction can be camed out directly on an absorbent body used to obtain DNA samples from surfaces and in particular trace DNA samples, without requiring any purification or isolation step This technique minimizes sample handling, reducing the risk of contamination.

Furthermore, it ensures that the entire amount of trace DNA (i.e. the maximum available) is available for amplification and so the possibility of failure through lack of DNA is minimized. Although simple, it breaks the culTent dogma of trace sample extraction and purification.

As used herein, the expression "non-biothgical surface" refers to a non-living surface such as that of an inert object or structure. Such surfaces yield environmental samples that are generally not replete with nucleic acids as is found in samples, generally known as "reference samples" that are taken directly from a biological organism such as a human. Samples obtained from non-biological surfaces will contain traces of nucleic acid only as a result of contact with or contamination by a biological organism. Thus, such surfaces tend to yield only trace DNA samples, in particular if they have been only briefly in contact with a biological organism such as an individual in particular the skin thereof or suspect as occurs in forensic analysis, or have been contaminated with other organisms such as bactena or viruses as may occur in hygiene or monitoring applications. When used in forensics, the non-biological surface from which trace DNA samples are recovered is suitably an inert surface such as the surface of an object obtained at a crime scene. However, the method may be applied to other sm-faces where in particular only trace DNA samples are recoverable associated with other fields such as hygiene monitoring in particular in environments in which food is prepared, or in military applications.

As used herein, the expression "trace DNA samples" refers to very small generally unquantifiable amounts of DNA that fall below recommended thresholds at any stage of an established technique.

The absorbent body is suitably formed of a fibrous material that may be synthetic or natural. Particular examples of materials that may form the absorbent body include nylon including flocked nylon, rayon, polyester, cotton, cellulose and nitrocellulose. Suitably the absorbent body is the headltip of a small swab, such as a neonatal swab, or a fragment of an absorbent membrane such as a cellulosic disc. The absorbent body is not required to be pre-treated with reagents such as reagents that lyse cells, denature proteins and/or preserve nucleic acids. Thus the body is suitably free of any such reagents. The use of such simple bodies such as on swabs or untreated membranes such as filter papers will minimise the costs of the procedure.

The body is suitably small enough to fit inside a conventional reaction vessel used to carry out an amplification reaction, such as a 200 p1 tube used for a conventional PCR. The body may be submerged in the amplification reaction mixture during the method, and consequently in such an embodiment the volume of the amplification reaction mixture should be at least sufficient to allow for submersion of the absorbent body. In one embodiment, the absorbent body is a head/tip of a swab, and the method comprises submersion of the head (or tip) of the swab in the volume of the amplification reaction mixture. Submersion, rather than simply saturation of the absorbent body/head of the swab, further promotes solubilisation of any collected material. Suitably therefore, the absorbent body will be less than 4 mm in diameter or width in at least one direction to allow it to be positioned in such a tube. Neonatal swabs are particularly preferred for use in the method of the invention because they appear to be extremely efficient at collecting trace DNA and are fortuitously sized (diameter of 2 mni, and length of8 mm) to fit perfectly into a standard 200 p1 PCR tube which may serve as the reaction vessel. They were designed initially for microbiological sampling from newborn infants and are not used in DNA forensics.

However, other swabs may be employed and suitably small sections cut from them after the trace DNA sample has been coflected so as to allow them to fit into the reaction vessel. However, in a favoured embodiment the amplification reaction mixture is applied to the whole of the absorbent body, rather than sections of the absorbent body, so that 100% of sample (including nucleic acid) collected enters the

S

reaction vessel. By using the whole of the absorbent body it is possible that all (100%) nucleic acid collected could be amplified. There is also less opportunity for cross contamination, especially from the operator, for example during the cutting of sections from the absorbent body, especially since the cutting imp'ement, such as scissors, would likely be contaminated. If the absorbent body, or headltip of a swab, needed to be separated from any support for the absorbent body, or rod of the swab, then cutting could occur on the support/rod. rather than on the absorbent body or headltip. In one embodiment the support or rod could be designed to be weakened at a particubr point so that the absorbent body or headitip could be easily removed by simply snapping the support/rod, rather than cutting.

Where the absorbent body is a fragment of an absorbent membrane, it is suitably in the form of a disc of diameter less than 4 mm, for example about 2 mm in diameter. The membrane is suitably less than 200 pm thick for example about 180 jtm thick. The membrane will be porous and have a pore size that is sufficient to absorb trace DNA, for instance a pore size of from 5-25 pm and suitably about II Mm. A particular examp'e of such a membrane is Whatman® grade I filter paper but other membranes will be usable.

In genera', the absorbent body is sdected so that it has a liquid capacity of less than SOpl, for instance from I to 20M1 such as from 2 to JOlt]. Preferably, the volume of amplification reaction mixture applied to the absorbent body exceeds that which is required to saturate the absorbent body so that there is at least some free liquid in the reaction vessel during the amplification reaction. However, the amount of the excess can be kept small, for example to ensure that there is from 2-30 M1 free liquid only in the reaction vessel. Since the reagents used in amplification reactions and in particular PCR amplification mixtures are expensive, this ensures that the method of the invention can be carried out in an economical and cost effective manner.

In this respect, the use of membranes and in particular small cellulosic discs may be a preferred embodiment. For instance, 2 mm diameter cellulose discs (cut from filter paper) were tested as an alternative sampling medium in place of neonatal swabs. They performed extremely well in small volumes of PCR reagent. The neonatal swab method requires 50 III of PCR reagent, while a single 2mm disc can be placed in lOul of PCR reagent and successfully amplified. This reduces the amount of PCR reagent used for each trace assay by 5 fold. In view of the cost of these reagents, this reduction has a significant financial benefit for forensics companies who carry out tens of thousands of these trace DNA tests a year.

However, the applicants have found that in some instances, the use of swabs and in particular neonatal swabs may be preferred where very low copy number DNA may be present as the results obtainable using such bodies may be more sensitive and may be more readily subject to enhancement carried out after amplification as discussed further below.

The absorbent body may be wetted prior to being contacted with the inert surface so as to maximize retrieval of trace DNA. Wetting may be effected by applying water or a suitable wetting buffer and in particular a controlled amount of water to the absorbent body. The amount added will depend upon the size of the absorbent body, but generally 1-2 j..tl water or wetting buffer will be sufficient.

Alternatively, the absorbent body may be immersed in water or wetting buffer pnor to use, and after removal from the solution, excess water/buffer is suitably shaken off before application to the surface.

In one embodiment the wetting buffer comprises a surfactant or detergent to aid solubility of any material collected from the non-biologica' surface. The presence of a surfactant or detergent in the wetting buffer however also decreases the chance of any inhibition of the amplification reaction by contaminants collected during contact with the non-biological surface. Upon app'ying the amplification reaction mixture to the absorbent body, comprising the wetting buffer, the surfactant or detergent will combine with the amplification reaction mixture to provide a buffer capable of counteracting the inhibition effect of contaminants. The surfactant may for example be a non-ionic surfactant, such as polysorbate, Triton® surfactants. and Tween® surfactants. The percentage of surfactant in the wetting buffer may be between 0.05% and 5% and preferably between 0.1 to 1% to provide particularly effective wetting buffers for collection from a non-biological surface. The applicant has also found that an aqueous solution of the detergent SD.S is an effective wetting buffer for collecting material from non-biologica' surfaces, in particular 0.5% to 10% aqueous SDS solutions, and most particu'arly 1% to 2% aqueous SDS solutions. Wetting buffers that can aid solubility of any material on non-biological surfaces, especially any nucleic acid material, are particularly favoured for use in the method of the present invention.

The wetting buffer may also compnse an additive capable of stabilising the amplification reaction, such as bovine serum albumin (BSA) or glycerol. The wetting buffer could for example comprise between 1% and 15% glycerol. preferably between 3% and 8% glycerol, and ahernatively or additionally BSA at a concentration of 500 to 5000 tg/ml, and preferably between 1000 and 2500 tgIml.

The Applicant has found that a wetting buffer containing DMSO in addition to water can improve the amount of material collected from a non-biological surface, and in particular approximately equal proportions of DMSO and water, such as 50 % of each, can double the amount of material collected from a non-biological surface over use of water alone.

Contact of the absorbent body with the surface is suitably by way of a swabbing or wiping action, which is easy when using a small swab such as a neonatal swab. Where the absorbent body is a fragment of a membrane, this is suitably applied to the surface using a suitable holding device such as tweezers which are used to press the fragment against the surface before being lifted off.

The step of contacting the absorbent body with the non-biological surface to recover nucleic acid thereon may comprise a preliminary step in the method of the invention.

In order to maximize the amount of nucleic acid collected on the absorbent body, it has previously been considered that the area of the surface that is swabbed or wiped with the absorbent body should be maximized. However, the applicants have found that using the method of the invention, small surface areas may be accurately sampled. for example areas of less than 1 cm2 and preferably less than 0.25 cm2 may be contacted or swabbed with the absorbent body and successful results still obtained.

This microsampling' technique, in which small areas of the surface are swabbed individually, may be preferable in certain applications as this provides for high resolution' sampling. Such a microsampling' technique may for example be undertaken through use of a neonatal swab (with the head/tip of the swab of dimensions typically of 2 mm in diameter by 8 mm in length), rather than the larger swabs commonly used for swabbing surfaces. A method comprising submersion of a neonatal swab (or swab of similar dimensions) in the amplification reaction mixture is a particularly advantageous combination, improving the chances of solubilisation of collected material from the small head. By analyzing individual areas in this way, the nsk that some specific nucleic acids become masked' by other nucleic acids, present in higher quantities in the general area is minimized. The method of the invention makes this possible because no nucleic acid is lost from the absorbent body after sampling and therefore it is of a higher sensitivity. For instance, if keys of a mobile phone are swabbed individually, and the resultant absorbent bodies analysed individually also, nucleic acids from occasional users of the phone may be determined against the background of nucleic acid from a dominant user. This is because the Ukelihood is that an occasional user would have made firm contact with a limited number of specific keys on the mobile phone, and thus these will form more concentrated sample of skin cells containing nucleic acid of that individuat Once the absorbent body has been contacted with the surface, it is suitaNy placed into the reaction vessd immediately. The reaction vessel may already contain an amplification reaction mixture or this may be introduced into the vessel after the absorbent body.

The applicants have found that by using small absorbent bodies, amplification reactions can be achieved in spite of the presence of levels of inhibitors that may be found in the environment. Conventional amplification reactions can be carried out including those that involve thermal cycling such as polymerase chain reaction (PCR), ligase chain reaction, NASBA, LUXIM or PlexorTM amplification reactions as well as isothermal nucleic acid amplification reactions such as strand displacement amplification (SDA), transcription-mediated amplification aMA), loop-mediated isothermal amplification (LAMP), rolling circle DNA amplification, and multiple displacement amplification. As used herein, the expression "amplification reaction mixture" refers to a partic&ar combination of reagents that are required in order to effect a particular amplification reaction. The amplification reaction mixture used in each case and the conditions applied to effect the amplification are well known in the art. For instance the amplification reagent may comprise one or more sets of primers to ensure that a particular target nucleic acid is subject of the amplification reaction, as well as enzymes, nucleotides, probes, salts and buffers that may also be required to effect a particular amplification reaction.

Suitably however, the amplification reaction mixture used in the reaction contains at least one additive that can stabilize the reaction and in particular enzymes used in the reaction. Thus, in the case of PCR for instance, the amplification reagent will suitably contain at least one of bovine serum albumin BSA) or glycerol. The additive may however be present in the wetting buffer, which is indeed favoured since the additive will have a longer period of time to associate with material collected from the non-biological surface. The additive may be present in both the amplification reaction mixture and the wetting buffer to further improve the stabilisation.

In addition or alternatively, the amplification reagent may further comprise a reagent that can lyse any cells that are present in the environmental sample. Suitable ytic reagents are detergents and in particular, non-ionic surfactants such as polysorbate, for instance polysorbate 20, sold under the trade name "Tween" and Triton-X 100. The detergent may however be present in the wetting buffer, which is indeed favoured since the detergent will have a longer period of time to associate with the material collected from the non-biological surface, and especially more time to provide lysis of any cells and more chance to decrease the chance of inhibition of the amplification reaction by contaminants, before contacting the amplification reaction mixture. The detergent or surfactant may be present in both the amplification reaction mixture and the wetting buffer to further decrease the chance of any inhibition of the amplification reaction by contaminants collected during contact with the non-biological surface.

Inclusion of such reagents enhances the ability of the reaction mixture to operate even in the presence of inhibitors found in the environment and so to operate as "direct amplification" reagents. As used herein, the expression "direct amplification reagent" refers to a reagent that is able to amplify nucleic acids that have not been subject to substantial purification procedures. In particular. it refers to amplification reaction mixtures that comprise buffers that will allow an amplification reaction to proceed in the presence of at least some inhibitory compounds.

In a particular embodiment the amplification reaction is a polymerase chain reaction. Thus the direct amplification reagent is a polymerase chain reaction (PCR) reagent comprising the elements necessary for canying out such a reaction including primers, nucleotides, salts such as magnesium or manganese salts, a thermostable polymerase such as Taq polymerase and a buffer.

In particular the buffer is selected so that it will allow the PCR to proceed in the presence of inhibitory compounds. Buffers of this type are described in US2O1O/0015621. In particular, the buffer comprises at least 0.2%-0.9% polysorbate such as polysorbate 20. 3%-8% glycerol, and 1000-3000 pg/mi BSA.

In addition, the direct amplification reagent will further comprise 10-50mM Tris-FICI (pH 8.3), 30-80mM KCI, 1.4-2.4mM MgCl2. 0.01 %-0.04% Sodium azide, 100-350 pM of each dNTPs. and 0.10-0.35 U/pl of DNA polymerase.

Suitably, the said volume of amplification reagent is less than 200 p for instance from 40 to 100 pl such as about 50 pI in line with conventional PCR technology. Ideally the volume is as low as practically possiNe, consistent with saturating the absorbent body, typically 2-20 p1 used in the sampling.

Nucleic acid amplified during the method of the invention maybe detected in a conventional way, which may be either in situ in the reaction vessel or it maybe analysed separately. Thus amplified DNA maybe run on a gel and detected for instance using a dye, on the gel. Preferably however, amplification is detected in situ in one of the many well known homogenous assays in which amplification of nucleic acid is detected using a visual signaling system. Such systems generally rely on the use of dyes such as fluorescent dyes. These dyes are included in the reaction system in such a way that the visible signal they produce changes as an amplification reaction proceeds.

This change can be monitored using conventional monitoring equipment as an indicator that amplification has occurred. In some instances, continuous monitoring of the progress of the ampfification can be used to quantify the amount of DNA present.

These methods may be genenc methods, which include an intercalating dye whose fluorescence changes in the presence of double stranded nucleic acid and can therefore the change of fluorescence can be utilized to detect a bulk increase in nudeic acid present in a reaction mixture, indicative of a successful amplification reaction.

However, strand specific methods that utilize labeled probes or primers and optionally also intercalating dyes are also well known. Such methods include the TaqMan®, LUX®, PlexorTM and ResonSense® methods well known in the art. The presence of the absorbent body in the reaction vessel does not unduly impede the generation and detection of visible signals such as fluorescent signals that are associated with these methods.

In a particular embodiment, the contents of the absorbent body are suitably extracted from the absorbent body after amplification but prior to detection of nucleic acid. Surprisingly, this technique has been found to enhance the sensitivity of the results obtained, possibly because more of the amplified nucleic acid is released from the absorbent body for detection. The ease of application of this enhancement technique will depend to sonic extent upon the nature of the absorbent body. For instance, when the absorbent body is a swab such as a neonatal swab, the contents of the swab can be readily extracted after the amplification reaction using for example a centrifugation filter. The swab may be washed and the washings similarly collected and added to the contents of the swab for detection purposes. This surprising finding also indicates that the absorbent body. or head of a swab, may also protect the amplification reaction from inhibition by contaminants, and may further aid the amplification reaction, especially since absorbent bodies are often manufactured from similar materials used to manufacture amplification reaction vessels: the absorbent body would thus heat at a similar rate, and to a similar extent, during the thermal amplification processes.

The use of different dyes associated for instance with different primer sets, allows the reaction to be multiplexed so that more than one specific nucleic acid sequence may be detected in the same reaction, again as weB known in the art.

In particular, the DNA may be subject to analysis such as DNA profiling analysis. In such cases, the amplification reaction used in the method is designed to amplify one or more genetic loci containing a short tandem repeat (STR) sequences within human DNA. Generally, up to 30 for instance 16 primer pairs are used in such analysis in order to amplify 16 genetic loci. The presence of nucleic acid corresponding to each of these loci may be determined using an electropherogram in a conventional manner. The presence or absence of nucleic acids corresponding to the various STRs at the loci and the relative concentrations of these can provide an indication of the DNA profile of one or more individuals who have contributed to nucleic acid that has been taken up by the absorbent body.

An example of a commercial reagent able to carry out such analysis is Applied Biosystems® (AB) Identifier® Direct PCR master mix reagent.

According to a further aspect of the invention there is provided a kit for carrying out a method as described above which comprises an absorbent body as described above and at least one reagent that is useful in an amplification reagent. Suitably the kit contains a complete amplification reaction mixture in sufficient volume to carry out the method in accordance with the invention. The kit may comprise a nucleic acid amplification reaction vessel with a lid, wherein the lid is adapted to incorporate the absorbent body.

Such a lid may enable the absorbent body to contact, or be submerged in, the amplification reaction mixture upon closing/sealing the reaction vessel with the lid.

Such an adapted lid could further minimise the risk of contamination with nucleic acid and environmental contaminants from the operator performing the method. The kit may comprise an absorbent body attached to a support, such as a swab comprising a headJtip and a rod, wherein the support. or rod of a swab, is weakened at a particular point so that the absorbent body or head/tip can be easily removed by simply snapping the support/rod, rather than cutting, which process could introduce contaminants to the amplification reaction mixture. As illustrated hereinafter, the method of the invention is extremely useful and can eliminate DNA purification steps generally undertaken in DNA analysis. Different types of absorbent body maybe preferred in different situations that may be summarised as follows: * The use of a cellulose disc as the absorbent body in the method of the invention is quick, very cheap, and works well for heavily contaminated areas.

It can reduce PCR reagent costs by 5 fold, and eliminate DNA purification steps. It has been tested successfully on three mobile phones (10 replicates for each).

* The use of a neonatal swab as the absorbent body in the method of the invention is also very quick, cheap and works well on medium to heavily contaminated areas. It eliminates DNA purification steps and is extremely easy to use. It has been tested on four mobile phones successfully (10 replicates on three, 20 on the fourth) * The addition of the enhancement technique, in particular when used in conjunction with a neonatal swab as the absorbent body in the method of the invention is quick, cheap and is sensitive down to low-copy number levels. It can detect DNA from poor shedders, and can detect down to single copy levels of reference DNA in multiple tests. It has been tested on five mobile phones (3-6 replicates on four, ten replicates for one). It tends to generate frill profiles that can be confidently matched to individuals known to have touched the object. It eliminates DNA purification, and users only require 30 minutes of training before being competent in the new technique.

The invention will now be particularly descnbed by way of example and by reference to the accompanying drawings in which: Figure I shows a range of swabs including a large flocked swab used for conventiona' trace DNA forensics (far left) compared with a small neonatal flocked swab that is particularly suitable for use in the method of the invention; Figure 2 shows the results of an electropherogram showing a mixed (two person, one male, one female) DNA profile recovered from a 1cm square region of a BlackBerry® mobile phone, using the method of the invention; Figure 3 shows the results of an electropherogram showing a strong DNA profile recovered from a small 4 mm x 4 mm square region of a BlackBerry® mobile phone, using the method of the invention; and Figure 4 shows the results of electropherogram obtained using a method of the invention in which a 4 mm x 4 mm area of a mobile phone screen with a 2 mm diameter cellulose disc was used as the absorbent body.

Example 1

Method using swab as absorbent body A 1 cm2 region of a BlackBerry® mobile phone was swabbed using a wetted neonatal flocked swab. After swabbing. the flocked nylon tip was cut off the swab with scissors and placed direcdy in a 200 tl PCR tube. (A conventional flocked swab would be too large to fit into such a tube.) To the tube containing the neonatal swab tip was added Applied Biosystems® (AB) Identifiler® Direct PCR master mix reagent (50 tl). Approximately 25 jJ of the reagent was absorbed into the swab tip, with 25J remaining free in the tube.

The tube was then subject to a conventional PCR cycling conditions in accordance with the manufacturer's instructions. After amplification. DNA was separated and visualized by capillary electrophoresis. The results are shown in Figure 2. These results showed a very clear mixed (two persons, one male, one female) DNA profile indicating that two individuals had handled the phone.

The method was repeated using 21 individual swabs taken from various areas of the phone. As the neonatal swabs were small (2 x 8 mm), single keys on the phone could be swabbed independently with ease. Out of the swabs taken, 14 of the 21 gave results that contained alleles for more than 6 loci and would therefore be likely to be sufficient for matching to a suspect. 8 of the 21 swabs gave results containing all 16 loci used for analysis. The ALT key on the phone (approximately 5 mm square) gave a clear, strong, full 16 loci profile that matched the owner of the phone exacfly at all oci.

Example 2

Method using celtulosic disc as absorbent body A 4mm x 4mm region (0.16cm2) of a BlackBerry® mobile phone was swabbed using a 2mm diameter cellulose disc wetted with 1 p1 of distilled water, held in fine forceps. After swabbing the disc was transferred directly to a 200 p1 PCR tube.

To the tube containing the cellulose disc was added 10 J of Applied Biosystems® (AB) Identifiler® Direct PCR master mix reagent. Approximately I tl of the reagent was absorbed into the disc, with 9 p1 remaining free in the tube.

The tube was then subject to a conventional PCR cycling conditions in IS accordance with the manufacturers instructions. After amplification, DNA was separated and visualized by capillary electrophoresis. The resulis are shown in Figure 3. These results showed a very clear profile from a single male individual.

The method was repeated using 8 additional samples taken from various areas of the phone. The volume of PCR reagent was repeated twice at 10 iii, three times at 25 pl and three times at 50 pl. As the cellulose discs were small (2mm diameter). top and bottom sections of single keys on the phone could be swabbed separately with ease. Out of the samples taken, 5 of the total 9 samples gave results that contained the full 16 loci profile of the owner of the phone. Best results were obtained from the lOul volume of PCR reagent (3/3 samples giving full 16 loci profiles), while the results from the 50 p1 volume of PCR reagent were poor (0/3 samples giving 16 loci profiles).

The 25 p1 volume of PCR reagent was intermediate (2/3 samples giving full i6 loci profiles). This indicates the benefit of reducing the PCR volume, which causes an effective increase in target DNA concentration.

The experiment was repeated a further three times with an additional three mobile phones. In all cases it was possible to rapidly recover the DNA profile of the owner of the phone using the 2 mm celiulosic disc, 10 tl PCR volume method.

Example 3

Further use of cellulosic discs as absorbent body 2 mm diameter discs were punched from a sheet of Whatman® grade 1 180 pm thick cellulose filter paper. The discs were wetted with I tl of sterile distilled water, and used to swab a 4 mm x 4 mm area of the screen from a Blackberry® mobile S phone. After swabbing, the disc was transferred using forceps to a 200 p1 PCR tube containing 10 p1 of Applied Biosystems® (AB) Identifier® Direct PCR master mix reagent. A 28 cycle PCR amplification was carried out as is usual PCR amplification of human DNA, and the PCR product analysed on an Applied Biosystems® 3500x1 capillary electrophoresis instrument in accordance with standard protocols. Three independent repeat samples showed strong, full DNA profiles that exactly matched the user of the mobile phone (example result shown in Figure 4).

To replicate this result, an additional three mobile phones from different users were tested using the same method. Each phone was sampled 10 times using 2mm cellulose discs, giving a total of 30 tests. In each case the tests generated sufficiently good DNA profiles to identify the user of the phone. Success rates were dependant on whether the user of the phone was a high or low shedder of DNA. Samples taken from the phone belonging to a user known to be a high shedder showed strong DNA profiles in 10/10 replicates. Samples taken from a medium shedder showed good DNA profiles in 5/10 replicates. Samples taken from a poor shedder showed usable profiles in 3/10 replicates.

The results show that using a cellulose disc as the absorbent body in the method of the invention appears most useful as a method to sample trace DNA from items that are likdy to be reasonably heavily contaminated i.e. items that are touched regirlarly. It may be less suitaNe to detect low shedders, which would typically be found using low template/low copy number DNA analysis. The cellulose disc method could however bring a significant cost saving by reducing the amount of PCR reagent needed.

Example 4

Increased sensitivity by enhancement step Three mobile phones belonging to different users were tested using the neonatal swab method described in Example 1, taking 10 swab samples from each phone. The technique worked well on two of the three phones, generating DNA profiles that could be confidently matched to the DNA profile of the user of the phone (phone I gave 6/10 positive swabs, phone 2 gave 7/10 positive swabs). The third phone gave weak results, and it was not possible to confidently match any profiles to the user of the phone. This result was likely due to the user of this phone being a poor shedder of DNA.

An enhancement step was developed whereby the PCR reagent was removed from the swab after amplification and placed in a Centricon® centrifugal filter concentration column. The swab was rinsed three times with a 150 tl volume of distilled water, removing the l5Opl rinse solution to the same Centricon® colunm.

These three wash steps extract PCR product that was trapped inside the swab, to a total volume of 500 tl. The Centricon® column was then centrifuged at 14000 g for 30 minutes, and the remaining retained liquid (between 10-20 tl) captured in a new tube.

1 p.1 of this liquid was loaded onto an Applied Biosystems® 3500x1 capillary electrophoresis instrument in accordance with standard manufacturer's protocols.

This enhancement technique was tested on the previously mentioned 10 repeats of the neonatal swab PCR reactions from a phone belonging to a poor DNA shedder.

None of these results showed a sufficiently strong profile to match them to the phones user prior to enhancement. After the enhancement described above, 8/10 samp'es gave DNA profiles that were suitable for comparison with the owner of the phone.

The technique greatly increased the sensitivity of a DNA profiling, to levels equivalent to low template or low copy number profiling. It is hypothesised that as the sample is captured by the neonatal swab it is absorbed into the body of the swab, and the majority of amplification takes place within the swab, rather than the free Uquid in the PCR tube. Some PCR products will escape the swab into the free liquid, but much remains within the swab. This enhancement step ensures all PCR product is washed from the swab, collected and concentrated for analysis.

Example 5

Testing of enhanced method using reference DNA The sensitivity of this enhanced neonatal swab technique was tested using standard, known concentrations of reference DNA. A sample of reference DNA (9947A, Applied Biosystems®) was diluted to give a range of DNA concentrations.

Neonatal swabs were wetted with sterile distilled water, and reference DNA was added to the tip of the swab in a 1 lii volume. Three repeats (i.e. three independent swabs) were carried out for the following amounts of reference DNA amounts per swab: 200 pg. 100 pg, 50 pg. 25 pg. 12.5 pg, 6.25 pg and 3.125 pg. All swabs were subjected to a 28 cycle amplification in 50 R1 of PCR reagent previously described, and were then subjected to the enhancement step described above. lp.l of product from each swab was loaded onto an Applied Biosystems® 3500x1 capillary electrophoresis instrument in accordance with standard protocols. The DNA profiles generated were compared against the known DNA profile of the 9974A DNA standard, and the number of correct alleles detected in each dilution step was noted. A summary of these results is

shown in Table 1.

Table 1. Number of alleles reported after neonatal swab and enhancement step.

The reference DNA used has a maximum of 26 detectable alleles in this test. At least 24 alleles were detectable if more than 25 pg DNA was present. This is equivalent to approximately four or more copies of human genorne.

DNA pg) Genome AlIdes reported (max = 26) copies (2n) Repeat 1 Repeat 2 Repeat 3 32.5 26 26 26 16.2 26 24 26 8.1 26 F 26 Ic 41 Ic I -r. I.J 13 2.1 9 23 17 6 1.0 20 0 17 3 0.5 16 5 10 The results in TaHe I suggest the neonatal swab plus enhancement step allow DNA detection well below lOOpg of input DNA. These levels are usually regarded as low template' or low cope number'. No drop-in' alleles were noted in the low DNA amounts used in this test, probably due to the relatively low cycle count (28 cycles) used. The neonatal swab direct PCR plus enhancement is a remarkably sensitive, and a very easy way to carry out trace DNA analysis. It is possible that the structure of the swab enhances the PCR reaction, perhaps increasing efficiency due to interactions of the DNA or polymerase with the nylon fibres of the swab.

Example 6

Testing of enhanced technique on test samples To further test the method, an additional four mobile phones were tested using the neonatal swab plus enhancement. Between 4-6 samples from S x 5 mm areas were taken from each phone by staff that had only received 30 minutes training in the technique. These samples were all processed as descnbed in Example 4 above, and the majority gave strong profiles. The resulting trace DNA profiles varied between phones; some were purely derived from the main user of the phone. and some were mixed profiles that contained alleles from both the main user of the phone and other individuals that had handled the phone. The resultant trace DNA profiles were used to calculate the probability of inclusion (using the Random Man Not hicluded method; RMNE) of the main user of the phone. The RMNE calculation results were expressed in the form of Likelihood ratios (LR). discounting any LR thwer than lxi 06. These results are shown in Table 2.

Table 2. Likelihood ratios expressing the likelihood of a given user contributing DNA to an observed profile derived from enhanced neonatal swabs samples taken from four mobile phones, compared with a random individual selected from the population. A likelihood ratio (LR) of I x io indicates that 1 in every I billion individuals selected at random from the population would be identified as a contributor to the mixture in the same way as the user. LRs lower than I x 106 are discounted (indicated by -in the table). In many cases the derived profile is a full DNA profile that is identical to the user of the phone.

Tube Area of phone Phone Likelihood ratio of contribution to the observed no. sampled user: mixed DNA profile for user: 1 2 3 4 I Callbutton I ---- 2 Call-end button 1 6.2 x i0'° --- 3 Microphone area 1 4.3 x io' --- 4 Speaker 1 4.9x 101 --- Left hand side 1 7.1 x io' --- 6 Right hand side 1 3.1 x 1016 --- 7 Onloffkey 2 -lOx io' -- 8 Call-end button 2 -lOx 101 -- 9 Volume button 2 -5.3 x 10 -- NumberOkey 2 -8.2x i09 -- II Speaker 2 ---- 12 Call-answer button 2 ---- 13 Call-answerbutton 3 --2.Ox lo9 - 14 Call-end button 3 --- Speaker 3 --8.3x l0' - 16 On/offbutton 3 --2.0x 10'° - 17 Centre of screen 3 --1.1 x - 18 Lockbutton 3 --8.3x 10' - 19 Home button 4 ---2.7 x l0' Centre of screen 4 ---7.7 x iO'3 21 Volume button 4 ---2.7 x l0' 22 On/off button 4 ---1.1 x io1° The results in Table 2 show that the enhanced neonatal swab method allows confident matching of trace DNA profiles to the users of phones, with no false positives reported. This demonstrates trace DNA detection to low copy number levels.

using a rapid. simple technique. It was notable that although the staff that carried out this experiment were experienced users of the 3500x1 instrument, none had ever carried out trace DNA sampling before, and received on'y 30 minutes training in this new technique.

Claims (15)

1. Claims I. A method for detecting the presence of a nucleic acid in the presence of environmental contaminants on a non-biological surface, said method comprising applying to an absorbent body that has been in contact with the non-biological surface, a volume of an amplification reaction mixture that is at least sufficient to saturate the absorbent body in a reaction vessel; calTying out an amplification reaction in said vessel, and detecting amplified nucleic acid produced.
2. 2. A method according to claim 1, wherein the absorbent body is wetted with a wetting buffer comprising a detergent or surfactant.
3. 3. A method according to claim 1 or claim 2, wherein the absorbent body is wetted with a wetting buffer comprising an additive capable of stabilising the amplification reaction.
4. 4. A method according to claims 1 to 3 wherein the contents of the absorbent body are extracted from the absorbent body after the amplification reaction and amplified nucleic acid present in extracts is detected.
5. 5. A method according to any one of claims ito 4 wherein the absorbent body is the head/tip of a swab such as a neonatal swab.
6. 6. A method according to claim 5 wherein the head/tip of the swab has dimensions of about 2 mm in diameter by 8 mm in length.
7. 7. A method according to any one of the preceding claims wherein the amplification reaction mixture used in the reaction contains at least one additive that can stabilize the reaction.
8. 8. A method according to any one of the preceding claims wherein the amplification reaction mixture further comprises a lytic reagent that can lyse cells.
9. 9. A method according to any one of the preceding claims wherein the amplification reaction mixture comprises a direct buffer that comprises at least 0.2%-0.9% polysorbate, 3%-8% glycerol, and 1000-3000 ig/ml BSA.
10. 10. The method according to claim 9 wherein the direct amplification reaction mixture further comprises, 10-50 mM Tris-HCI (pH 8.3), 30-80 mM KCI. 1.4-2.4 mM MgCI2.

    0.01 %-0.04% Sodium azide, 100-350 uM of each dNTPs, and 0.10-0.35 Ufttl of DNA polymerase.
11. 11. The method according to any one of the preceding claims wherein the volume of amplification reaction mixture is less than 200 jJ.
12. 12. The method according to any one of the preceding claims wherein the area of the surface that has been contacted with the absorbent body is less than 1 cm2.
13. 13. The method according to any one of the preceding claims which further comprises the step of contacting the absorbent body with the non-biological surface to recover nucleic acid thereon.
14. 14. A kit for carrying out a method according to any one of the preceding claims which comprises an absorbent body and at least one reagent that is useful in an amplification reagent.
15. 15. A kit according to claim 14 wherein the kit compnses a swab such as a neonatal swab, and the absorbent body is the head/tip of the swab.