Device for collecting biological material from a biological trace

Abstract

A device for collecting biological material, characterized in that it comprises a rod having, at one end, a dry absorbent body having a surface area of between approximately 1 and approximately 3.14 mm.sup.2, said absorbent body comprising surfactants and denaturing agents. The present invention also relates to the use of said device for sampling biological material from a biological trace having any surface area greater than a micro surface area of around 1 mm.sup.2 or having any volume greater than a micro-volume of around 1 microliter, and to a method for collecting biological material and a method for collecting and analyzing and/or identifying biological material, comprising a step of putting said device in contact with said biological material.

Claims

1. A device for collection of biological material, characterized in that it comprises a rod having at least at one end a dry absorbent body of an area of from 1 to 3.14 mm.sup.2, said absorbent body comprising surfactants and denaturing agents.

2. The device according to claim 1, characterized in that said end having said absorbent body is scored or ejectable.

3. The device according to claim 1, characterized in that said absorbent body is selected from the group consisting of cellulose, cotton, flocked synthetic fibers, hydrophilic polymers, fiberglass, Polytetrafluoroethylene and a porous ceramic.

4. The device according to claim 1 characterized in that said biological material is selected from the group consisting of physiological body fluids, cells or cell suspensions of humans or animals, in particular a contact trace, blood, bone marrow, buccal cells, semen, saliva, cervical fluid, hair, hair, skin, muscle, serum, synovial fluid, cerebral spinal fluid, lymphatic fluid, vaginal cells, urine, feces, and more generally, any human or animal biological tissue degraded or not; physiological body fluids or cell suspensions of plants; liquid products, extracts or suspensions of bacteria, fungi, plasmids, parasites, or viruses; liquid extracts or homogenates of human or animal body tissue; media from a nucleic acid synthesis; and mixtures of nucleic acids synthesized chemically or biochemically.

5. The device according to claim 1 characterized in that it comprises a hermetic case.

6. A method of collecting biological material, characterized in that it comprises a step of contacting the device according to claim 1 with said biological material.

7. The method for collection of biological material according to claim 6 characterized in that when the biological material to be collected is wet, the collection is performed directly, and when the biological material is in dry form, said process comprises a prior step of humidification of said absorbent body of the device or the humidification of said biological material with sterile water and/or pyrogen free.

8. The method of claim 6, wherein the method comprises contacting a biological trace having any surface from a micro-area of the order of 1 mm.sup.2 or having any volume, from a micro-volume of 1 microliter.

9. The method of claim 6, wherein the biological material is a human sample.

10. The method of claim 9, wherein the human sample is obtained by contacting a human with the device.

11. The method of claim 10, wherein the contacted human is not alive.

Description

(1) The accompanying drawings illustrate the invention:

(2) FIG. 1A represents, in cross-section, the collection device of the invention, outside its case. The gripping and handling area is at one end and the absorbent body is at an opposite end.

(3) FIG. 1B represents the collection device in its case, before or after collection of the biological material with an absorbent body in the rectilinear extension of the rod.

(4) FIG. 2 shows the collection device with an absorbent body (referred to as collection surface) having an angle with respect to the straight rod to facilitate collection, and its sealed case identifiable by a bar code.

(5) FIG. 3 shows a variant of the collection device or a second a collection device presenting a second absorbent body (referred to as a collection surface for preservation) is coupled to the first device as shown in FIG. 2. The gripping area is located in the middle of the rod and not at its end.

(6) FIG. 4A is a photograph of the device with an absorbent body of a surface of 3. 14 mm.sup.2, composed of nylon fibers flocked on the rod.

(7) FIG. 4B is a photograph of the device with an absorbent body having a surface of 1.13 mm.sup.2, composed of flocked nylon fibers on the rod.

(8) FIG. 4C is a photograph of a standard FloqSwab 4N6 Crime Scene Cat 3509C, Regular tip in plain tube swab, marketed by the Copan company.

(9) FIG. 5 is the composition of the IdentiFiler Kit as marketed in 2015.

(10) FIG. 6A is the comparison of the average fluorescence intensities obtained from 1 microliter of collected blood using a cellulose disk (to the left) and a standard swab (right).

(11) FIG. 6B is the comparison of the average fluorescence intensities obtained from 5 microliters of collected blood using a cellulose disk (to the left) and synthetic fibers flocked on a rod (to the right).

(12) FIG. 6C is the comparison of the average fluorescence intensities obtained from 1 microliter of semen collected using a cellulose disk (to the left) and synthetic fibers flocked on a rod (to the right).

(13) FIG. 7 is a photograph of 2 drops of blood of 5 microliters taken with the device according to the invention (surface 1.13 mm.sup.2).

(14) FIG. 8 is a fingerprint of blood taken from a standard swab and with the device according to the invention (surface 1.13 mm.sup.2).

(15) FIG. 9 photographs a microliter blood trace present in the barrel of a firearm and taken with the device according to the invention (surface 1.13 mm.sup.2).

(16) Other features and advantages of the invention may appear in the following examples, and are given purely by way of non-limiting illustration.

EXAMPLES

Example 1

Determining the Optimal Surfaces-Matrix of Cellulose

(17) In order to test the effect of the size on the collection efficiency, analysis and identification of DNA, it has been carried out at the following test:

(18) A dry solid absorbent body consisting of a dry cellulose matrix comprising FTA-type surfactant and denaturant agents (GE company) or NucleiCard (COPAN company), in the form of a disk (referred to as biological pellet) of a diameter of 1.2 mm (i.e. a surface of 1.13 mm.sup.2), 2 mm (i.e. a surface of 3.14 mm.sup.2), 3 mm (i.e. a surface of 7.06 mm.sup.2) and a square of 3.3 cm on the side (i.e. a surface of 10 mm.sup.2) were tested.

(19) In the absence of an industrial collection device, which is an object of the present invention, namely said absorbent body attached to a rod, the biological pellet is manipulated using a tweezers type clamp. It is noted that the users have a difficulty of use not allowing the use to be imagined, in particular by police forces on crime scenes. The experimental collection method is decomposed in the following manner: collecting the biological trace by wiping the support using horizontal or circular movements, or by simple buffering depending on the nature of the surface to be wiped.

(20) Tests have been performed on different types of traces likely to present DNA in varying amount and quality but mainly degraded. To this end, the collections have been made on so-called contact traces (wiping of different media such as the interior of a vehicle or daily objects), blood traces, buccal cells and diverse biological tissues (body elements in advanced degradation states such as putrefaction or carbonization) with each time a 1 microliter sample. The absorbent body is used dry without wetting the trace or absorbent body prior to collection.

(21) After collection, the absorbent body is brought into contact with one STR (Short Tandem Repeat) type DNA fragment amplification reaction medium such as identification Identifiler plus and Globalfiler (Lifetechnologies) kits and then inserted into the thermocycler for PCR.

(22) Thus, without delay, at the end of collection, the DNA trapped in the biological pellet is directly amplified by PCR (Polymerase Chain Reaction) in a thermocycler (GeneAmp PCR system 9700), without adding an additive to stabilize the amplification reaction, to determine human genetic profiles. This amplification reaction is carried out using commercial kits for amplification of STR-type DNA fragments (Short Tandem Repeat) such as Identifiler plus and Globalfiler kits (Lifetechnologies) according to the recommendations of the provider. Thus, in the case of use of Identifiler plus, the thermocycler is used with 28 PCR cycles and with Globalfiler with 29 PCR cycles.

(23) Genotyping of the PCR products is then performed using the ABI 3500 xl (Lifetechnologies) analyzer. Analysis of the results is performed using the GeneMapper ID-X version 1.2 Software

(24) The results obtained show that the surfaces of 10 mm.sup.2 and 7.06 mm.sup.2 do not allow to identify the DNA profile of the sample, matching the generally accepted idea by those skilled in the art that a microtrace DNA sample of the order of 1 microliter cannot be collected and analyzed with such collection devices.

(25) However, and this is a part of the technical solution of the present invention, the applicant has surprisingly and unexpectedly discovered that by further decreasing the surface area of the biological pellet, by performing the same tests with smaller absorbent body surfaces, of 1.13 to 3.14 mm.sup.2 it was possible to obtain a partial DNA profile (50% of the identified alleles) in the case of the surface of 3, 14 mm.sup.2 and even a complete DNA profile (100% of the identified alleles) with the surface of 1.13 mm2, against the technical prejudice of the person skilled in the art. Thus, the applicant has shown that it was not only possible to collect but also to analyze and accurately identify a microtrace of DNA of the order of 1 microliter, using a collection device.

(26) In order to validate the effectiveness of the rapid analysis process, biological traces have also been collected using a conventional swab device (FloqSwab, COPAN company) in amounts of 1, 3 and 5 microliters, and then analyzed with the aid of the Identifiler Plus, Identifiler Direct and GlobalFiler kits, according to the provider recommendations without pre-quantification of the DNA extracted from the entirety of the end of the swab.

(27) The results obtained from the rapid analysis process, subject of the present invention, are better than those obtained using the conventional analysis process, with a step of cell lysis and obligatory extraction (FIG. 6A). Indeed, the rapid collection and analysis system is mainly more efficient on the biological traces of very small areas and volumes (microtraces of the order of 1 mm.sup.2 and 1 l) due to the miniaturization of the collection surface and the absence of loss of genetic material (due to the absence of DNA extraction from the collection device).

Example 2

Determining the Optimal Composition of the Absorbent BodyCellulose Disk/Flocked Synthetic FiberSurface 1.13 mm.SUP.2

(28) Comparative performance tests are performed between an absorbent body composed of a cellulose disk of 1.13 mm.sup.2 comprising denaturing and surfactants agents and synthetic fibers of a total surface of 1.13 mm.sup.2 flocked on a rod and comprising these same denaturing agents and surfactants.

(29) Two types of traces are collected:

(30) A blood trace from 5 l of blood on a glass blade (FIG. 7)

(31) A semen trace from 1 l of dried semen onto a glass blade.

(32) Directly after collection, the absorbent bodies are deposited in the wells of a 96 well microplate containing 25 l of amplification reaction mixture of STR type DNA fragments (Short Tandem Repeat) provided with the GlobalFiler (Lifetechnologies) kit, then the microplate is inserted into a thermocycler (Gene Amp PCR System 9700) for the PCR reaction. Genotyping of the PCR products is then performed using the ABI 3500 XL sequencer analyzer (Lifetechnologies). The analysis of the reagents is carried out using the software GeneMapper ID-X version 1.2.

(33) The results allow to identify identical DNA profiles with cellulose and flocked synthetic fibers, namely an identification of 100% of the DNA profile of the blood and semen samples. In contrast, the flocked synthetic fibers device is more effective than cellulose disk device as the mean fluorescence intensity obtained from the genetic profile with the flocked fibers is greater than that obtained with the cellulose disc (FIGS. 6 B and 6C).

(34) In addition, a real comfort of use was noted with the flocked synthetic fibers version of the device, in comparison with the cellulose disk device manipulated using tweezers, especially when the trace to be collected is difficult to access as may be the case inside the barrel of a gun (FIG. 9).

Example 3

Verification of the Performance of the Flocked Synthetic Fibers Device of 1.13 mm.SUP.2 .and Treated with Denaturing and Surfactants Agents with Respect to a Standard Swab (4N6 FloqSwab, Marketed by the Company COPAN)

(35) Comparative efficacy tests are performed between the synthetic fiber with a total area of 1.13 mm2 flocked onto a rod and comprising denaturing and surfactants agents.

(36) Two types of traces are collected:

(37) 1. a thumbprint revealed on a glass

(38) 2. a bloody thumbprint revealed on the floor (tiles)

(39) In the case 1. The upper part of the thumbprint is collected with the device of flocked synthetic fibers while the lower part of the thumbprint is collected with a standard swab.

(40) In the case 2. wiping with the flocked synthetic fibers of 1.13 mm.sup.2 allowed to collect only the biological material materialized by two ridges papillary drawing due to the small area of the device. In contrast, 5 ridges were collected with standard swab altering especially the papillary drawing.

(41) Directly at the end of the collection, the absorption bodies are placed in the wells of a 96-well microplate containing 25 l of STR (Short Tandem Repeat) type DNA fragments amplification reaction mixture supplied with the GlobalFiler kit (Lifetechnologies), then the plate is inserted in a thermocycler (Gene Amp PCR System 9700) for PCR reaction. Genotyping of the PCR products is then performed using the analyzer ABI 3500 XL sequencer (LifeTechnologies). Analysis of reagents is carried out with the GeneMapper ID-X version 1.2 software. The sample taken by means of the rapid analysis method from flocked synthetic fibers of 1.13 mm.sup.2 on the papillary traces revealed on glass yielded a better result than that obtained using the method of conventional analysis with cell lysis step and required extraction. Indeed, a full DNA profile was obtained from the quick analysis process while a very partial DNA profile was obtained from the standard analysis process.

(42) Samples taken from the bloody thumbprint allowed to obtain a complete DNA profile to mean intensities of fluorescence equivalent between the two gathering and analysis devices. In contrast, the ability to collect only two peaks of the papillary drawing using the collection and rapid analysis device instead of 4 to 5 peaks with the collection and analysis device for an equivalent result demonstrates all the sensitivity and interest of the collection and rapid analysis device to limit the alteration of the trace (FIG. 8).