WO2017210748A1

WO2017210748A1 - Nucleic acid collection device and method

Info

Publication number: WO2017210748A1
Application number: PCT/AU2017/050577
Authority: WO
Inventors: Alexander William Walker; Jennifer TEMPLETON; Adrian Matthew Thornton Linacre
Original assignee: Flinders University Of South Australia
Priority date: 2016-06-10
Filing date: 2017-06-09
Publication date: 2017-12-14

Abstract

The present invention relates to devices for the collection of nucleic acid samples and methods for their use. Certain embodiments of the present disclosure provide a device for collecting a nucleic acid sample from an environment, the device comprising a collection head comprising a positively charged collection surface, wherein the collection surface comprises a saturation volume of between 1 ul and 100 ul, and a handle connected to the collection head.

Description

NUCLEIC ACID COLLECTION DEVICE AND METHOD PRIORITY CLAIM

[001] This application claims priority to Australian provisional patent application number 2016902269 filed on 10 June 2016, the content of which is hereby incorporated by reference.

FIELD

[002] The present invention relates to devices for the collection of nucleic acid samples and methods for their use. Particularly, in some embodiments, the present invention relates to devices and methods for the collection of forensic nucleic acid samples, such as touch DNA samples.

BACKGROUND

[003] Fingermarks are essential forensic evidence in numerous criminal investigations. Generating a DNA profile from a fingermark for the purpose of human identification would be beneficial in resolving a broad spectrum of criminal investigations, ranging from theft to crimes of violence.

[004] DNA retrieved from fingermarks deposited by touch (referred to as "touch DNA") is often degraded, limited in quantity and may contain elements that co-extract with the DNA, which can hinder subsequent analysis.

[005] Although forensic genetics has seen substantial improvements in DNA profiling sensitivity typically the use of less than 100 pg of DNA template (equating to -16 human somatic cells) can result in poor-quality profiles using standard forensic techniques. This limit of sensitivity still precludes many items that have been touched at a crime scene from generating a usable DNA profile, despite their potential importance in a criminal investigation; these samples can include triggers, steering wheels, bullet cartridges, and handles of knives. [006] In many criminal investigations, the ability to retrieve the maximum amount of DNA from samples such as touch DNA samples is of paramount importance in resolving the case. The first DNA profile generated from a fingerprint was reported more than a decade ago and revolutionized forensic science. Despite this advance in the field, research has found an extremely low success rate (5%-6%) when using the standard methodology in generating touch DNA profiles, highlighting the need for improvements to the touch DNA process.

[007] The standard workflow for touch DNA samples includes an extraction step before amplification. However, many current extraction processes are thought to result in a loss of DNA; hence a touch DNA swab initially containing less than 100 pg of DNA may result in insufficient template at the PCR step to generate an informative profile. Many touch DNA samples are therefore either not submitted for DNA typing or fail to generate any data without further enhancement. Previous attempts to generate DNA profiles from fingermarks used a low copy number (LCN) methodology. However, any enhancement of the amplification process from limited and low-level DNA has the inherent risk of introducing stochastic events such as allelic drop-out, allelic drop-in, or an increase in stutter heights or allelic imbalance for a heterozygote.

[008] In light of the above extraction-independent techniques for DNA analysis have been developed and these include "direct PCR" type methods such as described in Ottens et al. {Forensic Science International Genetics Supplement Series 4: e47-e48, 2013).

[009] Regardless of the choice of analytical methodology, optimizing the yield of DNA collected from an environment is critical when dealing with touch samples where limited quantities of DNA may be left behind. The number of cells transferred to touched objects is highly variable, and often results in less than 300 picograms of DNA. Most forensic short tandem repeat (STR) kits call for 0.5-1.0 nanograms of DNA (about 200 cells) for full profiles to be generated, thus it is crucial to maximize DNA recovery from handled objects.

[0010] As set out above, the success of a DNA profile depends largely on how much DNA is retrieved from the evidence. Cotton swabs, pre-moistened with distilled water, are used routinely in forensic laboratories. However, it has been recognized by the present inventors that these relatively large cotton swabs are not optimal for the collection of touch DNA samples.

SUMMARY

[0011] The present invention relates to devices for the collection of nucleic acid samples and methods for their use.

[0012] Certain embodiments of the present disclosure provide a device for collecting a nucleic acid sample from an environment, the device comprising:

a collection head comprising a positively charged collection surface, wherein the collection surface comprises a saturation volume of between lul and lOOul; and

a handle connected to the collection head.

[0013] Certain embodiments of the present disclosure provide a device for collecting a nucleic acid sample from an environment, the device comprising:

a collection head comprising a positively charged collection surface, wherein the collection surface comprises a saturation volume of between lul and lOOul; an engaging means to engage with a reaction vessel; wherein when the device and the reaction vessel are engaged via the engaging means, at least the collection surface of the device is positioned within the reaction vessel;

an ejection means comprising an ejection actuator that is actuatable by a user to eject at least the collection surface from the device into the reaction vessel when the device is engaged with the reaction vessel; and

a handle connected to the collection head.

[0014] Certain embodiments of the present disclosure provide a method for determining the presence of a nucleic acid in an environment, the method comprising: providing a device as described herein;

applying lOOul or less of a sample collection buffer to the environment and/or the device;

using said device to collect one or more nucleic acid samples from the environment; and

determining the presence or absence of more nucleic acids in one or more of said samples;

wherein the presence of a nucleic acid in one or more of the samples indicates the presence of the nucleic acid in the environment.

[0015] Other embodiments are disclosed herein. BRIEF DESCRIPTION OF THE FIGURES

[0016] Certain embodiments are illustrated by the following figures. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the description.

[0017] Figure 1 shows embodiments of a device comprising a substantially rigid handle with no actuator to eject the collection head from the handle. Panel A shows an embodiment of a device where the handle incorporates a negative angle bend. Panel A shows an embodiment of a device where the handle incorporates a positive angle bend.

[0018] Figure 2 shows an embodiment of a device comprising a substantially rigid handle and an ejection means comprising an ejection actuator proximate to the collection head of the device.

[0019] Figure 3 shows an embodiment of a device comprising a substantially rigid handle and an ejection means comprising an ejection actuator distal to the collection head of the device.

[0020] Figure 4 shows an embodiment of a device comprising a handle with a flexible or resilient portion and no actuator to eject the collection head from the handle.

[0021] Figure 5 shows an embodiment of a device comprising a handle with a flexible or resilient portion and an ejection means comprising an ejection actuator proximate to the collection head of the device. [0022] Figure 6 shows an embodiment of a device comprising a handle with a flexible or resilient portion and an ejection means comprising an ejection actuator distal to the

[0023] Figure 7 shows an embodiment of a collection surface that may be incorporated into any embodiment of a device wherein the collection surface comprises a split configuration such that the collection surface may be split into two or more fractions after ejection from the device.

[0024] Figure 8 shows an embodiment of a collection surface that may be incorporated into any embodiment of a device wherein the collection surface comprises a brush configuration.

[0025] Figure 9 shows an embodiment of a collection head that may be incorporated into any embodiment of a device wherein the collection surface comprises a removable cap which sits over the remainder of the collection head.

[0026] Figure 10 shows an example of a 200ul PCR tube as referred to herein. Panel A shows a top end view of a PCT tube. Panel B shows a side view of a PCT tube. All measurements shown are in mm.

[0027] Figure 11 shows an embodiment of a device with a sealing surface for contact with a reaction vessel.

[0028] Figure 12 shows an embodiment of a device with a sealing surface for contact with a reaction vessel in exploded view.

[0029] Figure 13 shows an embodiment of a device with a sealing surface for contact with a reaction vessel and engaged with a reaction vessel.

[0030] Figure 14 shows an embodiment of a dual sampling device.

[0031] Figure 15 shows an embodiment of a dual sampling device engaged with two reaction vessels. [0032] Figure 16 shows an embodiment of a dual sampling device in exploded view.

DETAILED DESCRIPTION

[0033] It has been recognised by the present inventors that relatively large cotton swabs as used in routine forensic practice are not optimised for the collection of touch DNA samples. In particular, it has been recognised by the inventors that the relatively large working volumes of these swabs, typically in the 100' s of microlitre range, may dilute touch DNA samples to the point that they are no longer suitable for forensic analysis. Moreover, it has also been recognised by the inventors that direct amplification of touch DNA off a swab is an optimal way of analysing touch DNA samples and, as such, the relatively large size of routine cotton swabs impedes the direct analysis of DNA as the swab head is generally too large and/or absorbent to be directly included in a reaction to detect a nucleic acid.

[0034] Accordingly, in a first aspect, the present invention provides a device for collecting a nucleic acid sample from an environment, the device comprising:

a collection head comprising a positively charged collection surface, wherein the collection surface comprises a saturation volume of between 1 ul and 100 ul; and

a handle connected to the collection head.

[0035] The device of the present invention is adapted to the collection of nucleic acid samples from an environment. Typically, nucleic acid samples collected from an environment are for later analysis to determine the source of the nucleic acid, such as is done in forensic analysis cases.

[0036] In light of the above, the "environment" referred to herein may be any environment in which a nucleic acid sample of interest may be found. For example, the environment may be a surface that has come into contact with a nucleic acid of interest such as a surface that has been touched by a person of interest or where a body fluid has been deposited on a surface. [0037] In some embodiments, the "environment" may also comprise an organism, or a cell, tissue or organ thereof, such as an animal, plant or microorganism; an organic product including a plant product such as timber or other plant material or an animal- derived product such as animal horns or ivory; a device used to collect a nucleic acid or organic sample; and the like.

[0038] In some embodiments, the device of the present invention has particular application in the collection of nucleic acid samples from an environment that has been touched by a person of interest and, as such, the device has particular application in the collection of nucleic acids from fingerprints left on a surface by a person of interest. In some embodiments, the present invention has particular application in the collection of a nucleic acid from environments such as adhesive tape, plastic bags, plastic covering of wires, plastics and metals associated with electric or electronic devices, metal surfaces of firearms or ammunition, natural substrates including plants and outer covering of animals.

[0039] The device of the present invention is particularly adapted to working with very low volumes of liquid as are typically used when attempting to collect DNA from a touched surface, referred to herein as 'Touch DNA' .

[0040] As such, in some embodiments, the collection surface comprises a saturation volume of less than 100 ul, less than 50 ul, less than 20 ul or less than 10 ul. In some embodiments, the collection surface comprises a saturation volume of: between 1 ul and 100 ul, between 1 ul and 50 ul or between 2 ul and 20 ul.

[0041] A "saturation volume" as referred to herein refers to the volume of liquid that the collection surface can absorb or adsorb before reaching a point of saturation, ie. the point at which no more liquid can be absorbed or adsorbed by the collection surface. Methods for determining saturation volume are known in the art.

[0042] Moreover, it is has been recognized by the inventors that it is highly beneficial for the collection surface (after collection of a nucleic acid) to be directly incorporated into a nucleic acid analysis technique without pre-processing, such as a DNA analysis technique. [0043] In some embodiments, it has been recognized that polymerase chain reaction ('PCR') may be a suitable analysis technique for touch DNA samples. As such, in some embodiments of the invention, it is desirable for at least the collection surface of the collection head of the device to be sufficiently small such that it can be fully immersed in the working volume of a PCR reaction in a PCR reaction vessel.

[0044] As such, in some embodiments, at least the collection surface of the collection head, when ejected from the handle, is sufficiently small such that it can be fully immersed in no more than 100 ul of liquid in a PCR tube; no more than 50 ul of liquid in a PCR tube; no more than 30 ul of liquid in a PCR tube; or about 25 ul of liquid in a PCR tube. PCR tubes may be of different sizes, but a commonly used PCR tube is a 200 ul PCR tube.

[0045] In some embodiments, at least the collection surface of the collection head, when ejected from the handle, is sufficiently small such that it can be fully immersed in no more than 100 ul of liquid in a 200 ul PCR tube; no more than 50 ul of liquid in a 200ul PCR tube; no more than 30 ul of liquid in a 200 ul PCR tube; or about 25 ul of liquid in a 200 ul PCR tube.

[0046] A person skilled in the art would readily recognize what is meant by a "200ul PCR tube". However, by way of non-limiting example, a "200ul PCR tube" includes the Applied Biosystems 200ul PCR tube illustrated in Figure 10. However, as would be appreciated by those of skill in the art, a range of other PCR tubes possibly having different dimensions are also contemplated by the present invention. For example, in some embodiments a "200ul PCR tube" may include a 200ul PCR tube comprising a screw cap closure rather than a friction fit closure as illustrated in Figure 10.

[0047] It will be appreciated that the collection head may be of any suitable configuration or shape. In some embodiments, the collection head is substantially spherical.

[0048] In some embodiments, the collection head comprises a volume of 125 mm or less, 64 mm 3 or less, 27 mm 3 or less, 8 mm 3 or less, or 1 mm 3 or less. In some embodiments, the collection head comprises a volume of at least 125 mm , at least 64 mm 3 , at least 27 mm 3 , at least 8 mm 3 , or at least 1 mm 3. In some embodiments, the collection head comprises a volume in the range of 1 mm 3 to 125 mm 3 , 1 mm 3 to 64 mm 3 , 1 mm3 to 27 mm 3 , 1 mm3 to 8 mm 3 , 8 mm3 to 125 mm 3 , 27 mm3 to 1235 mm3, 64 mm 3 to 125 mm 3 , 27 mm 3 to 125 mm 3 , or 64 mm 3 to 125 mm 3.

[0049] In some embodiments, the collection head comprises a substantially spherical head and comprises a size (diameter) of 5 mm or less, 4 mm or less, 3 mm or less, 2 mm or less, or 1 mm or less. In some embodiments, the collection head comprises a size (diameter) of at least 1 mm, at least 2 mm, at least 3 mm, at least 4 mm, or at least 5 mm. In some embodiments, the collection head comprises a size (diameter) in the range of 1 mm to 5 mm, 1 mm to 4 mm, 1 mm to 3mm, 1 mm to 2 mm, 2 mm to 5 mm, 2 mm to 4 mm, 2 mm to 3 mm, 3 mm to 5 mm, 3 mm to 4 mm, or 4 mm to 5 mm. In certain embodiments, the head has a different configuration or shape, and comprises a width, height and/or depth of one of the aforementioned sizes.

[0050] As set out above, the collection surface of the device of the present invention comprises a positively charged surface. In use, the collection surface of the device is contacted with a surface in the environment to be sampled in order to adhere nucleic acids from the environment to the collection surface. Without being bound by any particular mode of action, it is considered that negatively charged nucleic acids such as DNA or RNA are attracted to, and adhere to, the positively charged collection surface.

[0051] In light of the above, any suitable positively charged surface may be used for the collection surface. However, in some embodiments, the collection surface comprises a positively charged polymer or cationic polymer. In some embodiments, the collection surface may also comprise a natural or synthetic polymer, such as a polymer comprising an amide linkage or an amine or thiol group in the polymer backbone or side-chain. Suitable collection surfaces may be selected. Methods for producing a collection head comprising a positively charged collection surface are known in the art.

[0052] In some embodiments, the collection surface comprises Nylon. "Nylon" as used herein refers to a family of synthetic polymers that are typically made by reacting monomers such as lactams, acid/amines or stoichiometric mixtures of diamines (-NH2) and diacids (-COOH). Mixtures of these can be polymerized together to make copolymers. Nylon polymers can be mixed with a wide variety of additives to achieve many different property variations. "Nylon" as referred to herein should be understood to not refer to any one particular form of nylon, but refer to any member of the nylon polymer family that carries a net positive charge.

[0053] The collection surface may be any suitable configuration to promote transfer of a nucleic acid from an environment to the collection surface.

[0054] In some embodiments, the collection surface comprises a "flocked" surface. A flocked surface as referred to herein refers to a surface, which comprises a coating of particles or fibres (or similar) attached to a substrate. In some embodiments a flocked surface is made by applying a charge to the particles or fibres to be attached to the surface (ie. the flock) whilst the substrate is earthed. Flock material flies vertically onto the substrate attaching to previously applied glue. A number of different substrates can be flocked including textiles, fabric, woven fabric, paper, PVC, sponge, toys, and automotive plastic.

[0055] In some embodiments, the flocked surface of the present invention comprises positively charged polymer flock or nylon flock.

[0056] In some embodiments, the flocked surface comprises a flock fibre length of less than 0.6 mm. In some embodiments the flocked surface comprises a flock fibre length of less than 0.4 mm.

[0057] In some embodiments, the collection surface may also comprise a smooth or textured surface, a brush configuration, a microfibre cloth, a collection of fibres, or a woven configuration.

[0058] As set out above, the device of the present invention comprises a handle. In use, the handle of the device is gripped by a user to manipulate the device such that at least the collection head contacts the environment to be sampled in order to effect transfer of nucleic acids from the environment to collection head. The handle may be in any suitable configuration to promote effective sampling. For example, the handle may be straight or may include one or more bends to place the collection head at a suitable angle for effective sampling relative to the handle.

[0059] In some embodiments, the handle may be substantially rigid that is, in use, the handle does not substantially bend or deflect from its original configuration.

[0060] However, in some embodiments, the handle may comprise a flexible portion. In some embodiments, the handle may comprise a resilient portion.

[0061] A "flexible portion" as referred to herein contemplates a portion of the handle that may be bent or positioned to a desired angle in order to generate and hold a desired angle between the handle and the collection head of the device.

[0062] A "resilient portion" as referred to herein contemplates a portion of the handle that may flex under force in order to transiently allow an angle to be formed between handle and collection head. However, in this case, when the force causing flexion of the resilient portion is removed, the resilient portion will return to its original resting position.

[0063] In some embodiments the flexible or resilient portion may comprise a part of the handle. In some embodiments the flexible or resilient portion may comprise substantially all of the handle.

[0064] In some embodiments, the handle is indirectly connected to the collection head. For example, the collection head may be connected to a shaft that is slidably connected to the handle.

[0065] In some embodiments, the device comprises a shaft connected to the collection head and the shaft is slidably connected to the handle. In some embodiments, the shaft is housed within the handle.

[0066] The handle, or a portion of the handle, may be manufactured from any suitable material or materials to achieve the desired level or rigidity, flexibility or resilience. For example, in some embodiments, flexible polystyrene (PS), copolymer polypropylene (PP), may be used in order to produce flexible handle portions. In some embodiments thermoplastic elastomer (TPE/TPU) may be used in order to produce a resilient handle portion. Where it is desired for the handle or a portion thereof to be substantially rigid, materials such as metals, wood or substantially rigid polymers (such as polycarbonate or polystyrene), carbon fibre or ceramic, among others, may be used.

[0067] In some embodiments, the device of the present invention comprises an ejection means for ejecting the at least the collection surface from the device.

[0068] Ejection of at least the collection surface from the device is useful where the collection surface needs to be transferred to another vessel, such as a vessel for analysis of a nucleic acid present on the collection surface after sampling an environment. In some embodiments, ejection of at least the collection surface may include ejection into a PCR

[0069] Ejection of "at least the collection surface" may include ejection of all or part of the collection surface alone from the device or, in some embodiments, include co- ejection of another part of the device such as all or part of the collection head and/or a portion of the handle.

[0070] In some embodiments, the ejection means comprises an ejection actuator that is actuatable by a user to eject at least the collection surface from the device.

[0071] Actuation of the ejection actuator may be via any suitable means such as pushing, pulling twisting, bending, squeezing, slicing and the like to effect ejection of at least the collection head from the device.

[0072] Actuation of the ejection actuator may effect ejection of at least the collection head in any suitable way, which would be readily ascertained by one skilled in the art. For example, actuation of the ejection actuator may displace a friction fit between the collection head or surface and the handle; may sever a join between the collection head or surface and the handle; may unscrew a threaded join between the collection head or surface and the handle; and the like. [0073] The ejection actuator may be located at any convenient location on the device. For example, in some embodiments, the ejection actuator is positioned on the device proximate to the collection head. In some embodiments the ejection actuator is positioned on the device distal to the ejection head.

[0074] In some embodiments, the device further comprises an engaging means to engage with a reaction vessel, wherein when the device and the reaction vessel are engaged via the engaging means, at least the collection surface of the device is positioned within the reaction vessel.

[0075] In some embodiments, the engaging means may be configured as a collar that engages with the opening of a reaction vessel to form a substantially liquid tight seal between the engaging means and the opening of the reaction vessel. Typically, the engaging means or collar on the device is positioned such that when a reaction vessel is engaged with the engaging means, at least the collection surface of the device is positioned within the reaction vessel.

[0076] Formation of a seal between the engaging means and the opening of a reaction vessel may be via any convenient mechanism including, for example, a friction fit engagement, a threaded engagement or the like.

[0077] In some embodiments, the ejection means comprises an ejection actuator that is actuatable by a user to eject at least the collection surface from the device into the reaction vessel when the device is engaged with the reaction vessel.

[0078] A "reaction vessel" as referred to herein may be any vessel in which a nucleic acid analysis process or method may be conducted, such as a vessel in which a PCR reaction may be conducted. One skilled in the art would readily identify vessels suitable for the conduct of a PCR reaction or other nucleic acid analysis method. However, by way of example, a suitable reaction vessel for a PCR reaction would include a typical 200ul volume PCR reaction tube, an example of which is illustrated in Figure 10. [0079] In some embodiments, the device is a pre -primed device for collecting a nucleic acid sample. For example, the device may be spring loaded so as to prime the ejection means for use.

[0080] The device of the present invention is useful for the collection of a nucleic acid from an environment.

[0081] A "nucleic acid" as referred to herein may include any type of nucleic acid that might be found in an environment. As such, in some embodiments, the nucleic acid may comprise DNA or RNA. Furthermore, the subject nucleic acid may be in any suitable form such as single stranded or double stranded.

[0082] In some embodiments, the nucleic acid comprises DNA.

[0083] The sampled nucleic acid may come from any suitable source such as an animal, a plant or a microorganism such as a bacterium or fungus. However, in some embodiments, the nucleic acid comprises a nucleic acid from an animal, including for example human DNA and/or RNA.

[0084] The nucleic acid may also be derived from any part of a cellular organism. As such, a nucleic acid as referred to herein may include genomic DNA, mitochondrial DNA, chloroplast DNA, viral DNA, DNA or RNA from an extrachromosomal element and the like. In some embodiments, the nucleic acid comprises genomic DNA.

[0085] In some embodiments, the device comprises a plurality of collection heads, for example two or more collection heads. In this embodiment, the device provides a dual or multiple sampling device for collecting a nucleic acid sample from an environment.

[0086] In some embodiments, the device comprising a plurality of collection heads may comprise a single or multiple ejection means. In this regard, the device may comprise a single ejection actuator that actuates one or more of the collection surfaces, or may comprise multiple ejection means comprising an ejection actuator to actuate a single collection surface. [0087] In some embodiments, the device comprising a plurality of collection heads may comprise a plurality of engaging means, the engaging means engaging with a single reaction vessel.

[0088] In certain embodiments, the device comprises a single handle. In certain embodiments, the device comprises a plurality of handles.

[0089] In some embodiments, the device comprising a plurality of collection heads comprises a single handle. In certain embodiments, the device comprising a plurality of handles collection heads comprises a plurality of handles.

[0090] In a second aspect, the present invention also provides a device for collecting a nucleic acid sample from an environment, the device comprising:

a handle connected to the collection head.

[0091] In a further aspect, the present invention also provides a method for determining the presence of a nucleic acid in an environment, the method comprising:

providing a device according to the first or second aspect of the invention; applying lOOul or less of a collection buffer to the environment and/or the device;

wherein the presence of a nucleic acid in one or more of the samples indicates the presence of the nucleic acid in the environment. [0092] In some embodiments, the nucleic acid contemplated in the second aspect of the invention may be any nucleic acid as described with reference to the first aspect of the invention.

[0093] An "environment" with respect to the second aspect of the invention may be any environment as described with respect to the first aspect of the invention.

[0094] As set out above, the method of the second aspect of the invention comprises applying lOOul or less of a collection buffer to the environment and/or the device. In some embodiments, the method of the second aspect of the invention comprises applying lOOul or less; 50ul or less; 20ul or less; lOul or less; between lul and 50ul; or between 2ul and 20ul of a sample collection buffer to the environment and/or the device.

[0095] In the method of the present invention, the collection buffer is a liquid that serves to facilitate the transfer of a nucleic acid from the environment to the collection surface of the device. As such, the collection buffer may be applied to the collection surface of the device, the environment, or both, to facilitate the transfer of a nucleic acid from the environment to the collection surface.

[0096] The collection buffer may be any suitable liquid for this purpose. For example, a range of collection buffers is described by Thomasma and Foran ( Forensic Sci 58(2): 465-469, 2013). Typically, the collection buffer is a solvent for a nucleic acid and thus includes, for example, liquids such as water and detergent in water solutions such as a solution of one or more of sodium dodecyl sulfate (SDS), Triton X, Tween 20, Formula 409, or Simple Green, Phosphate Buffered Saline (PBS), Ethanol (100%-70% v/v), Isopropanol (100% -70% v/v).

[0097] In some embodiments, where the collection buffer comprises a solution of a detergent, the detergent is dissolved in water at a concentration of less than 5% (v/v); less than 4% (v/v); less than 3% (v/v); less than 2% (v/v); less than 5% (v/v); less than 1% (v/v); less than 0.5% (v/v); less than 0.2% (v/v); or about 0.1% (v/v). In some embodiments where the detergent is SDS, the concentration may be about 2%. In some embodiments where the detergent is Triton X, the concentration may be about 0.1%.

[0098] In some embodiments, the collection buffer may also comprise a PCR facilitating reagent, for example: Betaine, DMSO, PEG, PCRboost, BSA or PrepNGo Buffer. In some embodiments, the PCR facilitating reagent may be added to the collection buffer before, during or after collection of a nucleic acid from an environment.

[0099] As set out above, the method of the present invention contemplates determining the presence or absence of more nucleic acids in one or more samples collected from an environment using the device of the first aspect of the invention. In this regard, any suitable method for detecting a nucleic acid in one or more samples may be used. For example, nucleic acids may be detected using methods which incorporate one or more of: polymerase chain reaction (PCR); Southern blotting, Northern blotting, isothermal nucleic acid amplification (eg. as reviewed by Gill and Ghaemi, Nucleosides, Nucleotides and Nucleic Acids 27: 224-243, 2008); optical detection methods utilizing a DNA binding dye; gel electrophoresis (including agarose and polyacrylamide gels); capillary electrophoresis; cDNA synthesis; mRNA detection; miRNA detection; and the like.

[00100] In some embodiments determining the presence or absence of more nucleic acids in one or more of said samples comprises a Polymerase Chain Reaction step.

[00101] In some embodiments, determining the presence or absence of more nucleic acids in one or more of said samples comprises a Polymerase Chain Reaction step without a discreet DNA extraction step to extract DNA from the device. Examples of such methods include, for example direct PCR detection methods as described by Ottens et al. (supra, 2013).

[00102] In some embodiments where the nucleic acid detection method comprises a PCR amplification step, particularly a direct PCR amplification step, at least the collection surface of the device is included in the Polymerase Chain Reaction step. That at least the collection surface of the device may be directly incorporated into the PCR reaction vessel with the PCR reaction reagents and be left in the reaction vessel during the PCR amplification step.

[00103] In some embodiments, determining the presence or absence of more nucleic acids in one or more of said samples comprises the use of a DNA profiling kit. DNA profiling kits are supplied commercially by a number of companies such as Thermo Fisher / Applied Biosciences and Promega Corporation. For example, Applied Biosystems DNA profiling kits include: AmpFfSTR™ Identifiler Plus PCR Amplification Kit (15 STR loci plus amelogenin), GlobalFiler™ Kits (26 loci), AmpFfSTR™ NGM™ PCR Amplification Kit, AmpFfSTR™ NGM Select™ PCR, Amplification Kit, NGM SElect™ Express Kit (all 16 STR loci). Examples of DNA profiling kits supplied by Promega corporation include: PowerPlex® Fusion Systems, PowerPlex® Y23 System, PowerPlex® ESX and ESI Fast Systems, PowerPlex® 21 System, PowerPlex® 18D System.

[00104] Methods for the use of DNA profiling kits would be readiliy ascertained by those skilled in the art. A range of methods for the use of DNA profiling kits are also detailed in the literature, for example: Holt et al., Journal of Forensic Sciences 47: 66- 96, 2002; Butler, Advanced topics in Forensic DNA Typing Methodology, Chapter 5 99-139, Academic Press, 2011; Mulero et al., Forensic Science International: Genetics Supplement Series 1: 121-122, 2008; Rao Coticone et al., Int J Legal Med 118: 224- 234, 2004.

[00105] The present disclosure is further described by the following examples. It is to be understood that the following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description. EXAMPLE 1 NUCLEIC ACID SAMPLING DEVICE WITH RIGID HANDLE

[00106] Embodiments of devices for sampling nucleic acids, which incorporate a substantially rigid handle, are shown in Figure 1.

[00107] Referring to Figure 1, devices 100, 120 each comprise a collection head 150 that comprises a positively charged collection surface 152. In these embodiments at least collection surface 152, and optionally all of collection head 150 is manufactured from a positively charged polymer such as nylon. In use, collection surface 152 is contacted with a surface in the environment to be sampled in order to adhere nucleic acids from the environment to collection surface 152. Without being bound by any particular mode of action, it is considered that negatively charged nucleic acids such as DNA or RNA are attracted to, and adhere to, the positively charged collection surface 152.

[00108] Collection surface 152 has a saturation volume of between 1 ul and 100 ul, that is the collection surface is able to absorb or adsorb between 1 ul and 100 ul of liquid. In some embodiments, , the collection surface has a saturation volume of between 2 ul and 20 ul of liquid. Typically, as described later, lOOul or less of a sample collection buffer is applied to either the environment and/or the device before, or while, the device is contacted with the environment in order to facilitate transfer of nucleic acids from the environment to collection surface 152.

[00109] Collection head 152 may have any suitable configuration to achieve a desired saturation volume. For example, collection head 152 may comprise a flocked surface, be configured as a brush, comprise a textured surface, or comprise a smooth surface. In some embodiments, collection head 152 may comprise a flocked surface or be configured as a brush. Where collection 152 head comprises a flocked surface, in some embodiments the flocked surface comprises a flock fibre length of less than 0.6 mm or less than 0.4mm.

[00110] Collection head 150 is adapted to be removed from device 100, 120 and sized such that it can be placed into a 200 ul PCR tube 162. The dimensional details of a 200 ul PCT tube are shown in Figure 10. Removal of collection head 150 from device 100, 120 may be by any suitable means. For example collection head 150 may be cut through the stem portion of collection head 150 or, as described later, collection head 150 may be ejected from a device of the present invention via an ejection means. In any event, collection head 150 is sized such that at least collection surface 152 is fully immersed in no more than 100 ul of liquid in a 200ul PCR tube and in some embodiments can be fully immersed in less than 50 ul, or less than 30 ul of liquid, in a 200 ul PCR tube.

[00111] Devices 100, 120 also each comprise a handle 140. In use, handle 140 is gripped by a user to manipulate device 100, 120 such that at least collection head 152 contacts the environment to be sampled in order to effect transfer of nucleic acids from the environment to collection head 152. Handle 140 may be any suitable configuration to promote effective sampling. For example handle 140 may be straight (not shown Figure 1) or may include one or more bends, such as those shown at 142, 144 to place collection head 152 at a suitable angle for effective sampling relative to handle 140. As will be appreciated by those of skill in the art, handle 140 may be configured in any way to place collection head 152 at a desired angle relative to handle 140.

[00112] Handle 140 may be manufactured out of any suitable material such as a polymer, metal, a plastic, wood or the like.

EXAMPLE 2 - EJECTION MEANS

[00113] Figures 2 and 3 show further embodiments wherein devices 200, 300 each further comprise an embodiment of an ejection means 220, 320 for ejecting at least the collection surface 152 from handle 140.

[00114] In this Example, where an illustrated part is numbered as per Example 1, the numbered part should be considered to function as described with reference to Example 1.

[00115] Referring to Figure 2, device 200 further comprises ejection means 220 for ejecting the collection head 150 from handle 140. Ejection means 220 comprises shaft 224 and actuator 222, wherein shaft 224 runs through aperture 226 in handle 140 and can make contact with collection head 150. Collection head 150 is held by friction between the stem of collection head 150 in aperture 226. When actuator 222 is depressed by a user, shaft 224 pushes the stem of collection head 150 out of aperture 226 and thus releases collection head 150, including collection surface 152, from handle 140. In some embodiments, collection head 150 may be released, for example into PCR tube 162.

[00116] Referring to Figure 3, device 300 shows a further embodiment of a device comprising actuating means 320 for ejecting at least collection surface 152 from handle 140. In this embodiment, handle 140 is substantially straight and indirectly connected to the collection head by shaft 324.. Ejection means 320 comprises shaft 324 and actuator 322. In this embodiment, shaft 324 runs through aperture 326 which runs substantially the full longitudinal length of handle 140 and can make contact with collection head 150. In device 300, actuator 322 is positioned on device 300 distal to collection head 150. This may be contrasted with ejection means 220 in device 200 where actuator 222 is positioned on the device more proximate to collection head 150.

EXAMPLE 3 - NUCLEIC ACID SAMPLING DEVICES WITH A FLEXIBLE OR RESILIENT HANDLE PORTION

[00117] In this Example, where an illustrated part is numbered as per Example 1 or 2, the numbered part should be considered to function as described with reference to Example 1 or 2.

[00118] Embodiments of devices that comprise a flexible or resilient portion in the handle of the device are shown in Figures 4 to 6.

[00119] Referring to Figure 4, device 400 comprises a resilient or flexible portion 440 in handle 140. Where portion 440 is a flexible portion, portion 440 may be bent or positioned to a desired angle in order to form and hold a desired angle between handle 140 and collection head 150.

[00120] Where portion 440 is a resilient portion, portion 440 may flex under force in order to transiently allow an angle to be generated between handle 140 and collection head 150, however, in this case when the force causing flexion of 440 is removed, portion 440 will return to its original resting position.

[00121] Figure 5 illustrates a device similar to that shown in Figure 2 with the change that handle 140 comprises a flexible or resilient portion as shown at 540.

[00122] Device 500 also further comprises engaging means 560 to reversibly engage with a 200ul PCR tube 162, wherein when engaging means 560 and the PCR tube 162 are engaged, at least collection surface 152 is positioned within PCR tube 162. In device 500, engaging means 560 is configured as a collar that engages with the opening of PCR tube 162 to form a substantially liquid tight friction seal between engaging means 560 and PCR tube 162. As would be appreciated, an engaging means similar to engaging means 560 could also be incorporated into other embodiments of the invention including any and all embodiments described herein such as devices 100, 200, 300, 400 and 600.

[00123] The use of an engaging means 560 to reversibly engage with the reaction vessel permits the collection head 150 to be ejected into the reaction vessel while the device is engaged with the device.

[00124] These features provide further advantages, particularly where it would be highly desirable to: (i) protect the collection head prior to sampling; (ii) readily release the collection head for sampling an environment; and (iii) be able to eject the collection head directly into a reaction vessel after sampling with no handling of the collection head (ie. via the ejection means while the PCR tube is engaged with the device).

[00125] Figure 6 illustrates a device similar to that shown in Figure 3 with the change that handle 140 comprises a flexible or resilient portion as shown at 640. In this embodiment, in order to accommodate movement in handle 140 at flexible or resilient portion 640, shaft 324 of ejection means 320 has a corresponding flexible or resilient portion 624. EXAMPLE 4 SPLIT COLLECTION HEAD

[00126] Figure 7 shows an alternative embodiment of collection head 150, illustrated in Figure 7 as 750. Alternative collection head 750 may be incorporated into other embodiments of the present disclosure, including, for example device 100, 200, 300, 400, 500 or 600.

[00127] Collection head 750 may be substantially as described herein with respect to collection head 150, with the change that collection head 750 comprises two parts 751, 752. A device comprising collection head 750 can be used to sample an environment for a nucleic acid substantially as described herein. However, when collection head 750 is ejected or otherwise removed from a device, collection head 750 may be split into two halves, which can be used to provide redundant or matched samples for analysis or archiving.

[00128] As would be appreciated by those skilled in the art, further embodiments of collection head 750 are also contemplated where collection head 750 comprises more than 2 parts, such as 3, 4 or more parts which would allow for further replicate samples to be taken from a single sampling device.

EXAMPLE 5 - ALTERNATE COLLECTION SURFACE CONFIGURATIONS

[00129] Figure 8 shows an alternative embodiment of collection head 150, illustrated in Figure 8 as 850. Alternative collection head 850 may be incorporated into other embodiments of the present disclosure, including, for example device 100, 200, 300, 400, 500 or 600.

[00130] Collection head 850 may be substantially as described herein with respect to collection head 150, with the change that collection head 850 comprises a collection surface configured as a brush comprising projections 860 and inter-projection spaces 862. A device comprising collection head 850 can be used to sample an environment for a nucleic acid substantially as described herein. [00131] Figure 9 shows an alternative embodiment of collection head 150, illustrated in Figure 9 as 950. Alternative collection head 950 may be incorporated into other embodiments of the present invention, including, for example device 100, 200, 300, 400, 500 or 600.

[00132] Collection head 950 may be substantially as described herein with respect to collection head 150, with the change that collection head 950 comprises a collection surface 952 that is detachable from collection head 950. In use as shown in panel A, collection surface 952 fits over collection head 950 and is held in place via a friction fit. After collection of a nucleic acid from an environment, as shown in panel B, collection head 952 may be detached from collection head 950 and placed into, for example, a PCR reaction vessel.

[00133] In another embodiment, the collection head may comprise fibres that are able to be detached from the collection head, and when removed may form a loose suspension in the reaction vessel that is then subject to analysis. A variety of methods for detaching the fibres from the collection head may be employed, including for example, detachment of the fibres upon action of an ejection means. It is envisaged that the increased spacing between the fibres after release and when in suspension should assist with amplifying the nucleic acid material released from the fibres.

EXAMPLE 6 - NUCLEIC ACID SAMPLING DEVICE WITH SEALING SURFACES

[00134] In this Example, where an illustrated part is numbered as per Example 1 or 2, the numbered part should be considered to function as described with reference to Example 1 or 2.

[00135] Embodiments of a device that comprise a sealing mechanism are shown in Figures 11 to 13.

[00136] Referring to Figure 11, there is shown a view of an embodiment of a device 450 with a sealing surface for contact with a PCT tube. The device 450 comprises a handle 140 with a flexible portion 440 which has a sealing surface 454 on the handle with in use contacts the internal surfaces of a PCR tube 162. The expanded area of the sealing surface 454 provides sealing surfaces which prevent PCR fluid contamination and evaporation and provide volumetric accuracy.

[00137] In addition, the device may further be a spring loaded and primed device, 450 typically for single use, and comprising a spring to hold the device in a configuration ready for sampling. The use of spring loading in the device is contemplated for embodiments of the device, including the devices 100, 200, 300, 400 500 and 600 as described herein.

[00138] Referring to Figure 12, there is shown an exploded view of an embodiment of a device 450 with a sealing surface for contact with a PCT tube. The device 450 comprises ejection means 320 comprising a shaft 324 and an actuator 322, the shaft having a flexible portion 624. The handle 140 comprises a flexible portion 440, and also comprises a sealing surface 454 on the handle with in use contacts the internal surfaces of a PCR tube 162. The expanded area of the sealing surface 454 provide sealing surfaces 454 which prevent PCR fluid contamination and evaporation and provide volumetric accuracy.

[00139] Referring to Figure 13, there is shown a view of an embodiment of a device with a sealing surface engaged with a PCT tube 162. The handle 140 does not comprise a flexible portion and comprises a sealing surface 454 with contacts the internal surfaces of a PCR tube 162. The sealing surface 454 prevent PCR fluid contamination and evaporation and provides volumetric accuracy.

EXAMPLE 7 - DUAL ASSAY DEVICE

[00140] In this Example, where an illustrated part is numbered as per Example 1 or 2, the numbered part should be considered to function as described with reference to Example 1 or 2.

[00141] Embodiments of devices that allow dual assay are shown in Figures 14 and 15. [00142] Referring to Figure 14, there is a view of an embodiment of a device 1050 comprising a dual/combined ejector means 1020, a handle 1040 for use with the dual ejectors means 1020 having an actuator 1022 and shafts 1024, and two collection heads 152 for ejection into PCR tubes 162. The device allows sampling onto two collection heads 152 which are in close proximity and then ejected into separate PCR tubes.

[00143] Referring to Figure 15, there is a view of an embodiments of a device 1050 comprising a dual/combined ejector means 1020, a handle 1040 for use with the dual ejectors means, engaged with PCR tubes 162.

[00144] Referring to Figure 16, there is shown an exploded view of an embodiment of a device 1050 for dual sampling. The device 1050 comprising a dual/combined ejector means 1020 comprising an actuator 1022 and shafts 1024, a handle 1040 for use with the dual ejectors means, and two collection heads 152 for ejection into PCR tubes 162.

EXAMPLE 8 - PROCEDURE FOR RECOVERING 'TOUCH' DNA FROM CASEWORK EXHIBITS

[00145] An example of a methodology for collecting a DNA sample from a fingerprint is set out below. As will be appreciated, such a procedure may be used for a variety of applications, including for example, forensic DNA sampling.

[00146] Moisten the tip of a nucleic acid collection device comprising a flocked nylon collection surface (as described herein) with 4 ul of Triton X (0.1 %), pre-heated to 70°C.

[00147] Hold the swab vertically over the item to be sampled, so that only the collection surface of the device is in contact with the item. Swab 10 strokes vertically (top-to-bottom direction) and 10 strokes horizontally (left-to-right direction) over a visible fingermark on the item. In this regard, a collection head size (swab size) of about 1.5 mm has been found to be perform well in recovery of nucleic acids. [00148] After swabbing, detach the collection surface from the device and place it directly into a 200ul PCR tube (pre-labelled with case details). The dimension details of a 200 ul PCR tube are shown in Figure 10.

[00149] Seal PCR tubes and send tubes to the pre-PCR set up lab for processing. Clean work areas (as detailed previously), change gloves and proceed to next sample.

[00150] Reference: "DNA profiles from fingermarks", J E.L Templeton and Adrian Linacre. Biotechniques 57: 259-266 (November 2014) doi 10.2144/000114227.Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the features referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the features.

[00151] Also, it must be noted that, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context already dictates otherwise.

[00152] Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.

[00153] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[00154] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential. [00155] Future patent applications may be filed on the basis of the present application, for example by claiming priority from the present application, by claiming a divisional status and/or by claiming a continuation status. It is to be understood that the following claims are provided by way of example only, and are not intended to limit the scope of what may be claimed in any such future application. Nor should the claims be considered to limit the understanding of (or exclude other understandings of) the present disclosure. Features may be added to or omitted from the example claims at a later date.

Claims

1. A device for collecting a nucleic acid sample from an environment, the device comprising:

a collection head comprising a positively charged collection surface, wherein the collection surface comprises a saturation volume of between lul and lOOul; and a handle connected to the collection head.

2. The device of claim 1 wherein the device further comprises an ejection means for ejecting at least the collection surface from the device.

3. The device of claim 1 wherein the collection surface comprises a saturation volume of less than 5 Out.

4. The device of claim 1 wherein the collection surface comprises a saturation volume of less than 20ul.

5. The device of claim 1 wherein the collection surface comprises a saturation volume of between 2ul and 20ul.

6. The device of claim 1 wherein at least the collection surface of the collection head, when ejected from the handle is sufficiently small such that it can be fully immersed in no more than lOOul of liquid in a 200ul PCR tube.

7. The device of claim 1 wherein at least the collection surface of the collection head, when ejected from the handle is sufficiently small such that it can be fully immersed in no more than 50ul of liquid in a 200ul PCR tube.

8. The device of claim 1 wherein the collection surface comprises a positively charged polymer.

9. The device of claim 8 wherein the collection surface comprises nylon.

10. The device of claim 8 or 9 wherein the collection surface comprises a flocked surface.

11. The device of claim 10 wherein the flocked surface comprises a flock fibre length of less than 0.6 mm.

12. The device of claim 10 wherein the flocked surface comprises a flock fibre length of less than 0.4 mm.

13. The device of claim 8 or 9 wherein the collection surface comprises a brush.

14. The device of claim 1 wherein the handle comprises a resilient portion.

15. The device of claim 1 wherein the handle comprises a flexible portion.

16. The device of claim 1 wherein the ejection means comprises an ejection actuator that is actuatable by a user to eject at least the collection surface from the device.

17. The device of claim 16 wherein the ejection actuator is positioned on the device proximate to the collection head.

18. The device of claim 16 wherein the ejection actuator is positioned on the device distal to the collection head.

19. The device of claim 1 wherein the device comprises an engaging means to engage with a reaction vessel, wherein when the device and the reaction vessel are engaged via the engaging means, at least the collection surface of the device is positioned within the reaction vessel.

20. The device of any one of claims 1 to 19 wherein the nucleic acid comprises DNA.

21. The device of claim 20 wherein the nucleic acid comprises human DNA or RNA. j 1

22. The device of claim 20 or 21 wherein the nucleic acid comprises genomic DNA.

23. A device for collecting a nucleic acid sample from an environment the device comprising:

a handle connected to the collection head.

24. A method for determining the presence of a nucleic acid in an environment, the method comprising:

providing a device according to any one of claims I to 23;

25. The method of claim 24 wherein 5 Out or less of a sample collection buffer is applied to the environment and/or the device.

26. The method of claim 24 wherein 20ul or less of a sample collection buffer is applied to the environment and/or the device.

27. The method of claim 24 wherein between 2ul and 20ul of a sample collection buffer is applied to the environment and/or the device.

28. The method of any one of claims 24 to 27 wherein determining the presence or absence of more nucleic acids in one or more of said samples comprises a Polymerase Chain Reaction step.

29. The method of any one of claims 24 to 28 wherein determining the presence or absence of more nucleic acids in one or more of said samples comprises a Polymerase Chain Reaction step without a discreet DNA extraction step to extract DNA from the device.

30. The method of claim 28 or 29 wherein at least the collection surface of the device is included in the Polymerase Chain Reaction step.

31. The method of any one of claims 24 to 30 wherein the nucleic acid comprises DNA,

32. The method of claim 30 wherein the nucleic acid comprises human DNA or RNA.

33. The method of claim 31 or 32 wherein the nucleic acid comprises genomic DNA.

34. The device of claim 1 or the method of claim 24 as hereinbefore described with respect to any one of the Figures and/or Examples.