The present invention is within the field of molecular biology. More closely it relates to a flocked swab for use in a method for collecting, preserving and transferring biological samples in trace amounts for subsequent molecular biology analysis.

Background of the invention

Long-term storage, transport and archiving of nucleic acids on filter paper or chemically modified matrices is a well-known technique for preserving genetic material before the DNA or RNA is extracted and isolated in a form for use in genetic analysis such as PCR. Thus, EP 1563091 (Smith et ol, Whatman) relates to methods for storing nucleic acids from samples such as cells or cell lysates. The nucleic acid is isolated and stored for extended periods of time, at room temperature and humidity, on a wide variety of filters and other types of solid support or solid phase media.

In the collection of nucleic acids for subsequent analysis, the challenge is to collect as much sample (e.g., DNA from a crime scene) as possible, to allow maximum yield available for amplification.

Within prior art sample collection has been done by swabbing using a flocked fiber swab but the recovery of sampled amounts are often low and may vary. When a DNA extraction process is used to extract DNA from the swab, % recovery is usually not as high as expected and can be variable.

US 6168922 describes a device and method for storing and purifying nucleic acids from a biological source, comprising and adsorbent material having impregnatead chaotropic salts. The nucleic acids are eluted from the adsorbent material while amplification inhibitors, such as proteins, from the biological sample remain bound to the adsorbent.

US 9040675 relates RSM (RNA Stabilisation Matrix) chemicals used on a solid matrix for the extraction and storage of nucleic acids from a sample, such as a biological sample wherein a composition comprising a protein denaturant, a reducing agent, a buffer, and optionally a free-radical trap or RNase inhibitor is present in the solid matrix in a dried format

However, methods and devices that integrate nucleic acid extraction, stabilization, and storage/preservation from a biological sample within a single process with high yield are still highly desirable and needed.

Summary of the invention

The present invention provides a device and method for sample collection of optimal amounts of nucleic acid for subsequent analysis. The invention provides a flocked fibre collection swab of sufficient volume to collect enough sample for subsequent nucleic acid analysis. The flocked swab is either pre-treated with a solution comprising chaotropic agent(s) or dipped in such a solution before sampling. The chemistry of the solution serves to increase the quantity of sample retrieved, and stabilise and protect genetic material from degradation.

The low recovery and variation within prior art, is increased/improved according to the present invention where the swab can be used directly in an amplification reaction, such as 'direct' PCR. Also, addition of chaotrope increases DNA yield.

The flocked swabs used in the invention are provided on a tip or head of solid shaft, such as molded plastic applicator shaft. The head or tip may vary in size and shape and is coated with short polyester, such as nylon, fibers that are arranged in a perpendicular fashion on the tip. The flocked swab is a specimen collection device with tufts of polyester material attached to one end of a plastic shaft; used to collect forensic and biological specimens such as blood, saliva, semen, skin, bone or hair cells, bacterial and viral pathogens. The flocked swabs allow a greater amount of specimen to be collected and remain on the surface of the fiber for complete elution in the transport, assay or processing medium. The complex "webbing" of flock fibers collect and maintain more specimen and they then release it faster and more completely than traditional fiber swabs.

The surface area of a flocked swab is increased compared to traditional spun fiber swabs, also the sample is more readily released. The shape of the head or tip may be spherical or teardrop. Sphere volume = 4/3 Pi r3, teardrop volume = 2/9 Pi r2 r3 h (where r is the radius of the spherical part, and h is the height of conical part].

In a first aspect, the invention relates to a method for collecting a biological sample, such as a forensic sample or a crime scene sample, the method comprising the steps of a) providing a swab including a handle wherein one tip or end of the handle is coated with fibres , such as polyester fibres, like nylon, to form an absorbent flocked head, wherein the head having a surface area of 3-80 mm ² , such as 3.14mm ² and 78.54mm ² (surface area calculated for a sphere) and that the swab head would fit into a small volume reaction well/tube, such as 10-200 pi, preferably 25-50 pi;

b) collecting a biological sample comprising nucleic acid on the head by contact between the head and the sample,

c) subjecting the nucleic acid in the sample to nucleic acid (NA) amplification.

The reason for the head size is that a small flocked swab is ideal for the collection of a small quantity of sample / trace evidence, such as touch DNA, skin cells, gunshot residue etc. Also, swab must be small enough to fit into 25ul PCR volume to allow 'direct' PCR, but of sufficient size to collect adequate quantity of NA through swabbing process.

The biological sample may be body fluid or tissue and may comprise a buccal swab, a saliva sample, a serum sample, a plasma sample, a feces sample, a urine sample, a spinal fluid sample, a sputum sample, a sperm sample, lymphatic fluid, or a suspension of cells or viruses.

The flocked head is preferably sphere or drop shape and has a diameter of 1-5 mm.

In one embodiment the flocked head is treated with a predetermined concentration of chaotropic agent(s).

In another embodiment, the flocked head is contacted with a solution with a

predetermined concentration of chaotropic agent(s) before collecting the sample. In this embodiment the head is wet before sampling In a further embodiment, the nucleic acid sample is contacted with a solution with a predetermined concentration of chaotropic agent(s) before adding the flocked head for collecting the sample

The chaotropic agents are preferably selected from chaotropic salt(s) comprising an iodide salt or a guanidinium salt, such as guanidinium salt comprises guanidine hydrochloride, guanidine isothiocyanate, or guanidine thiocyanate, and optionally RSM (RNA Stabilisation Matrix) chemicals: a protein denaturant, a reducing agent, a buffer, and optionally a free- radical trap or RNase inhibitor is present in the solid matrix in a dried format.

Preferably the concentration of the solution is 500 - 1500 mM Guanidinium thiocynate. Optionally, the solution further comprises 10 - 50 mM MOPS.

The invention enables collection of 10 pg- 1000 ng nucleic acid is collected in step b) of the method of the invention described above.

In one embodiment of the method the sample in step c) is not released before amplification. In another embodiment the sample in step c) is released before amplification.

In a second aspect, the invention relates to a collection swab comprising a shaft with a flocked head in one end, said head having a diameter of 1-6 mm and surface area between 3mm ² and 80 mm ² obtained by flocked polyester fibers provided on the head.

Preferably the collection swab has fibres that are coated with a chaotropic agent.

The coating is with an iodide salt or a guanidinium salt, such as guanidinium salt comprises guanidine hydrochloride, guanidine isothiocyanate, or guanidine thiocyanate, and optionally RSM (RNA Stabilisation Matrix) chemicals: a protein denaturant, a reducing agent, a buffer, and optionally a free-radical trap or RNase inhibitor is present in the solid matrix in a dried format. Preferably the concentration of the solution applied to the flocked head is 500 - 1500 mM Guanidinium thiocynate, preferably also comprising 10 - 50 mM MOPS.

Detailed description of the invention

The present disclosure generally relates to methods and devices that integrate the steps of nucleic acid extraction and stabilization from a biological specimen collected on a solid matrix. The nucleic acids are released from the devices (flocked swab) at the time of analysis thereof.

The sample collecting device of the invention has at least two advantages over current swab formats:

a. The swab head/fibres will either be pre-treated/coated reagents and preservative to stabilise and protect genetic material from degradation. Reagents would include a chaotrope such as Guanidinium thiocynate. Alternatively, a buffer containing a chaotrope could be applied to the swab or sample area prior to swabbing.

Experiments have demonstrated use of a buffer containing Guanidinium thiocynate and MOPS increased DNA yield by up to 30% when compared to uncoated swabs or application of water to the sample area. b. The swab head will be small enough to be used for 'direct' PCR, therefore the swab can be added directly to the PCR reaction, it will not need to undergo lengthy, inefficient extraction processes to remove the sample e.g., DNA from the swab. The whole sample will go into the amplification/PCR/STR reaction, therefore reducing loss of DNA, which could potentially be scarce if e.g., collected from a crime scene.

EXPERIMENTAL

The below Experiments describe preparation and use of a flocked swab for collecting, preserving and transferring trace samples for molecular biology.

Experiment 1.

Aim:

To evaluate optimal concentration of Guanidinium thiocynate / MOPS buffer (GITC buffer) to use in Powerplex 18D STR profiling reactions (Promega DC1808).

Methods:

A 100% GITC buffer was prepared, containing 2.538 M Guanidinium thiocynate and 72 mM MOPS, pH ~7.5. This was diluted and lul aliquots were used to spike Powerplex 18D STR profiling reactions containing 250pg or lng purified gDNA. Results:

The results are shown in Table 1 below. Table 1

Conclusions:

The present inventors have found an ideal range for optimal concentration of GITC buffer and gDNA concentration. The optimal range of GITC buffer concentration appears to be ~ 507.6 - 1269 mM Guanidinium thiocynate and 14.4 - 36 mM MOPS applied to the swab (and therefore added to the PCR reaction). The sampled gDNA concentration should be within 10 pg and 1000 ng.

Note: 60% GITC buffer was previously trialed (data not shown), this concentration inhibited the PCR reaction. lng gDNA:-

• Presence of GITC buffer (40 & 50%) appears to increase Peak Height (APH) compared to no GITC control.

250pg gDNA:-

• Presence of GITC buffer (40 & 20%) appears to increase Peak Height (APH) compared to no GITC control.

• 100% First Pass Yield obtained - 31 alleles were called for all samples. Experiment 2.

Aim:

To determine whether Guanidinium thiocynate / MOPS buffer (GITC buffer) improves DNA yield following recovery using small swabs suitable for 'direct' PCR. Two approaches were used, as described below.

Note: 100% GITC buffer contains 2.538 M Guanidinium thiocynate and 72 mM MOPS, pH ~7.5

Methods:

1. Swabs coated with GITC buffer and used to recover 'dried' QDNA:-

5ul GITC buffer was pipetted onto the tip of each swab head and was left to dry in a desiccator overnight. The following day, the swabs were used to collect lng gDNA that had been dried onto a surface (see below for details).

Note: gDNA collection involved swabbing the area where the gDNA was applied for 10 seconds.

IA. 5ul of 4% GITC buffer pipetted onto swab heads.

5ul of a 4% GITC buffer equates to -507.6 mM Guanidinium thiocynate and 14.4 mM MOPS applied to the swab (and added to the PCR reaction).

IB. 5ul of 8% GITC buffer pipetted onto swab heads.

5ul of an 8% GITC buffer equates to -1015.2 mM Guanidinium thiocynate and 28.8 mM MOPS applied to the swab (and added to the PCR reaction).

IC. No GITC control - uncoated swabs were used.

2. GITC buffer pipetted onto 'dried' qDNA sample & uncoated swab used to recover qDNA:-

5ul GITC buffer was dispensed onto lng gDNA that had been 'dried' onto a surface (*as described below). Uncoated swabs were then used to collect the gDNA.

Note: gDNA collection involved swabbing the area where the gDNA was applied for 10 seconds. 2A. 5ul water dispensed onto 'dried gDNA samples.

2B. 5ul of 4% GITC buffer was pipetted onto 'dried' gDNA samples.

5ul of a 4% GITC buffer equates to -507.6 mM Guanidinium thiocynate and 14.4 mM MOPS applied to the swab (and added to the PCR reaction).

2C. 5ul of 8% GITC buffer was pipetted onto 'dried' gDNA samples.

5ul of an 8% GITC buffer equates to -1015.2 mM Guanidinium thiocynate and 28.8 mM MOPS applied to the swab (and added to the PCR reaction).

2D. 5ul of 0.1% SDS solution was pipetted onto 'dried' gDNA samples.

3. Uncoated swabs stored in GITC buffer post sample collection:- 3A. uncoated swabs placed in 25ul 4% GITC post sample collection.

3B. uncoated swabs placed in 25ul 8% GITC post sample collection.

3C. uncoated swabs stored 'dry' post sample collection.

Swab details:

Custom 'flocked' swabs were provided by Puritan. Swab heads were ~2.7 mm x 5.4 mm in size (semi-sphere or spherical cap shaped) The flocking material was nylon.

NOTE: post gDNA collection, all swabs were cut with a scalpel to remove the lower ~l/3rd of the bud. This was placed directly into the PCR reaction.

*Preparation of lnq gDNA 'dried' samples:-

4ul aliquots of gDNA @250pg/ul (lng total gDNA per spot) were aliquoted onto Petri dishes and left to dry overnight (with the lid on).

Note: the base of the Petri dishes had small circles ~5mm in diameter drawn on them with a marker pen & gDNA was placed within the circle.

When swabbed - the swab was rubbed over the area of the circle for 10 seconds.

4. Experimental controls:

4A. Positive control: lng gDNA in 25ul PP18D STR reaction. 4B. lng gDNA in 25ul PP18D STR reaction + lul 20% GITC buffer

4C. lng gDNA in 25ul PP18D STR reaction + lul 40% GITC buffer

4D. lng gDNA in 25ul PP18D STR reaction + lul 20% GITC buffer + uncoated swab

4E. lng gDNA in 25ul PP18D STR reaction + lul 40% GITC buffer + uncoated swab

4F. NTC: NO gDNA in 25ul PP18D STR reaction.

Results

The results are described below in Table 2.

Table 2

Conclusions:-

Experiment 1:

• Swabs coated with 5ul of 4% GITC buffer collected 3X more DNA than uncoated swabs. Full STR profiles (i.e., 33 alleles) were obtained for all samples, however some heterozygous alleles produced Peak Height Ratio's (PHR's) below 0.6.

• Swabs coated with 5ul of 8% GITC buffer collected 2X more DNA than uncoated swabs, however full STR profiles were not obtained for the 4 samples tested.

Experiment 2:

· Application of 5ul 4% GITC buffer to dried gDNA prior to swabbing yielded ~8% more

DNA than use of water. Full STR profiles (i.e., 33 alleles) were obtained for all samples and all heterozygous alleles produced Peak Height Ratio's (PHR's) above 0.6. • Application of 5ul 8% GITC buffer to dried gDNA prior to swabbing yielded -21% less DNA than use of water.

• Application of 5ul 0.1% SDS to dried gDNA prior to swabbing yielded -11% less DNA than use of water.

Other observations:

• Addition of GITC buffer directly to STR reactions enhanced Average Peak Height (APH) by -6 - 15% (samples 4B & 4C)

• Addition of swabs directly to STR reactions decreased Average Peak Height (APH) by -40% - however, full STR profiles (i.e., 33 alleles) were still obtained.

As discussed in Experiment 1, there appears to be an ideal spot for optimal concentration of GITC buffer and gDNA concentration. The optimal range of GITC buffer concentration appears to be - 507.6 - 1269 mM Guanidinium thiocynate and 14.4 - 36 mM MOPS applied to the swab (and therefore added to the PCR reaction). This equates to lul of 20% - 50%

GITC buffer used in a PCR reaction (or 5ul of 4% - 10% GITC buffer used in a PCR reaction).