CN107354149B

CN107354149B - Kit for extracting trace DNA and extraction method

Info

Publication number: CN107354149B
Application number: CN201710764074.6A
Authority: CN
Inventors: 朱奇; 王灵敏; 廖传荣; 孙继文
Original assignee: Guangzhou Genephar Biotechnology Co ltd
Current assignee: Guangzhou Genephar Biotechnology Co ltd
Priority date: 2017-08-30
Filing date: 2017-08-30
Publication date: 2020-05-12
Anticipated expiration: 2037-08-30
Also published as: CN107354149A

Abstract

The invention belongs to the field of trace DNA detection, and particularly relates to a kit and a method for improving the trace DNA extraction rate of trace sample DNA detection, single cell DNA detection of tumor cells, germ cells and the like in trace biological detection material DNA detection, forensic trace DNA detection, medical detection and clinical diagnosis and the like, wherein the kit comprises the following reagents: extracting magnetic beads, lysis solution, proteinase K, washing solution, nucleic acid precipitation aid and eluent from DNA; the invention also discloses a method for extracting trace DNA by using the kit. The kit and the method improve the detection rate, accuracy and sensitivity of trace DNA, can realize automatic extraction, realize parallel detection of multiple samples, and save labor and time cost.

Description

Kit for extracting trace DNA and extraction method

Technical Field

The invention belongs to the field of trace DNA detection, and particularly relates to a kit and a method for improving the extraction rate of trace DNA applied to trace biological detection material DNA detection, forensic trace DNA detection, medical detection and clinical diagnosis trace sample DNA detection, single cell DNA detection of tumor cells, germ cells and the like.

Background

Trace DNA refers to DNA that is present in amounts as low as picograms. In the field of forensic physical evidence, the field of detection of trace sample DNA in medical examination and clinical diagnosis, and the field of detection of single cell DNA of tumor cells, germ cells and the like, the number of detected biological sample cells is small, and the extracted DNA is in trace level.

In criminal case site investigation, with the increasing awareness of criminal reconnaissance, the increasingly concealed and scientific criminal means, the traditional material evidence range such as blood, hair, saliva, fine speckles and other obvious material evidence is usually consciously destroyed, and the trace biological material is easy to be ignored, so that a case loses the powerful evidence for solving the case, and how to better utilize the trace biological material evidence on the site has very important significance for case reconnaissance.

The trace biological detection material has various types, theoretically, human body cells can be left when the trace biological detection material is contacted with a human body, skin is a direct organ of the human body contacted with the outside, and a large amount of epidermal cells fall off every day when a person carries out metabolism every moment. Therefore, exfoliated cells from human skin are the most important source of trace biological samples, and these are also called trace contact biological samples. The trace biological detection material belongs to the difficult biological detection material, and the common cases of criminal investigation are as follows: fingerprints, cotton gloves, underwear pieces, chewing gum, cigarette butts, touch objects, and the like.

Since the first report of trace contact material evidence DNA by van Oorschot et al in 1997, the material evidence is attracting more and more attention, and the amount of inspection is increasing, for example, the center for identification of material evidence in Ministry of public Security, more than four thousand DNA inspection materials are inspected every two years, wherein about 40% of the trace contact material evidence is the trace contact material evidence. At present, the trace contact material evidence only has a small amount of exfoliated cells or DNA, so that the specific part of a sample is difficult to judge by naked eyes and can only be cut blindly, and the extraction of the DNA is influenced by overlarge cut material or introduction of external pollution, so that the success rate of detection is low, and the trace contact material evidence needs to be repeatedly detected and becomes a rate-limiting factor of case detection.

Currently, methods for detecting trace amounts of DNA in the market include a Chelex100 method, a silica bead column method, a magnetic bead method, and the like. Before extracting trace amount of DNA by the Chelex100 method, the material evidence needs to be cleaned, but the cleaning process may cause further loss of the original trace amount of DNA sample, thereby increasing the difficulty of DNA extraction, and meanwhile, the method has long detection time and poor detection effect. The silica bead column chromatography method for extracting trace DNA has the advantages of complex operation, low sensitivity and incapability of realizing automatic extraction although the sample detection rate is higher than that of Chelex 100. Compared with a silica bead column-passing method, the magnetic bead method is simple and convenient to operate, the tube rotating times are few, pollution and loss are not prone to occurring, automatic extraction can be achieved, trace DNA is extracted by the magnetic bead method based on an automatic workstation at present, and the detection rate is lower than 50%.

Thus, there is a need for improvement in the art.

Disclosure of Invention

In view of the above, it is necessary to provide a kit for improving the extraction rate of trace DNA and a method for extracting trace DNA by using the kit.

The technical scheme of the invention is as follows:

a kit for extracting trace DNA, the reagent comprising:

extracting magnetic beads, lysis solution, protease K solution, washing solution, nucleic acid precipitation aid and eluent from DNA;

the concentration of the protease K solution is 5-10 mg/ml;

the lysis solution comprises: sodium dodecyl sulfate or sodium dodecyl sarcosinate with the final concentration of 1-10%, guanidinium isothiocyanate with the final concentration of 2-5 mol/L, Tris-HCL (pH 8.0) with the final concentration of 50-100 mmol/L, EDTA (pH 8.0) with the final concentration of 1-10 mmol/L, Trition X-100 with the volume percentage of 0.5-7% and soluble salt NaCl with the final concentration of 0.1-1 mol/L;

the washing liquid comprises: Tris-HCl with the final concentration of 5-20mmol/L and ethanol with the volume percentage concentration of 70-80%, wherein the pH value is 7.0-8.0;

the nucleic acid precipitation aid is 1.5% of acrylamide, and polyacrylamide generated by the acrylamide can form a net to enable nucleic acid in liquid in the centrifugal tube to generate coprecipitation.

The eluent comprises: Tris-HCl (pH 8.0) with a final concentration of 5-20 mmol/L; Tris-HCl (pH 8.0) is preferred at a final concentration of 10 mmol/L.

Further, the DNA extraction magnetic beads are silicon-based coated ferroferric oxide magnetic beads, and the structure from inside to outside is as follows: the magnetic microsphere core, the polystyrene sealing magnet ring and the silicon dioxide coating functional group layer; the functional group of the silica-coated functional group layer includes a carboxyl group and the like.

Furthermore, the diameter of the magnetic bead is 200-2000 nm, and the density of the functional group is 1-5 mu M/mg;

preferably, the magnetic beads have a diameter of 1000nm and a functional group (e.g., carboxyl group, etc.) density of 2.5. mu.M/mg.

Preferably, the concentration of the proteinase K solution is 10 mg/ml;

preferably, the lysis solution comprises: guanidine isothiocyanate with a final concentration of 3mol/L, sodium dodecyl sulfate with a final concentration of 2%, Tris-HCL with a final concentration of 50mmol/L (pH 8.0), EDTA with a final concentration of 10mmol/L (pH 8.0), Trition X-100 with a volume percentage of 2% and a soluble salt NaCl with a final concentration of 1 mol/L.

Preferably, the washing solution comprises: Tris-HCL with the final concentration of 10mmol/L, ethanol with the final concentration of 80 percent by volume percentage and pH8.0;

preferably, the eluent comprises: Tris-HCl at a final concentration of 10mmol/L, pH 8.0.

A method for extracting trace DNA by using the kit comprises the following steps:

(1) lysing the sample cells in a centrifuge tube;

(2) adding isopropanol into the mixed solution cracked in the step (1), blowing, beating and uniformly mixing;

(3) adding the extracted magnetic beads and the nucleic acid precipitation aid into the solution obtained in the step (2), and mixing;

(4) instantly centrifuging the mixed solution obtained in the step (3), and absorbing and discarding the liquid;

(5) adding a washing solution into the centrifuge tube in the step (4) for washing, sucking and removing the washing solution, and airing;

(6) adding eluent into the centrifuge tube in the step (5), fully shaking and uniformly mixing;

(7) and (4) instantly centrifuging the mixed solution obtained in the step (6), transferring the eluent into a new centrifugal tube for later use, or directly using the magnetic bead mixed solution added with the eluent in the step (6) for downstream PCR amplification or STR typing detection.

Further, a method for extracting trace DNA by using the kit specifically comprises the following steps:

(1) collecting trace biological detection materials: collecting according to different methods according to different detection materials, and sticking or wiping the fingerprint or the contact object with an exfoliated cell sticking device or a cotton swab; for the detection materials such as cotton yarn gloves, underwear fragments, chewing gum, cigarette butts and the like, samples with the size not more than 1cm multiplied by 1cm can be directly cut for detection.

(2) Loading the micro-biological detection material collected on site into a nuclease-free centrifuge tube, adding 250-500 mul of lysis solution, wherein the addition amount of the lysis solution can be properly selected according to the size of the centrifuge tube and the size of the detection material, and the optimal volume of the lysis solution is 250-500 mul; adding proteinase K (10mg/ml)

(3) Fully cracking the material evidence sample by using the high-salt environment of the cracking solution in the step (2) under the conditions of 2000rpm, 55 ℃ and 10-20 min (preferably 20min), releasing the material evidence exfoliative cell genome DNA, and then adding isopropanol with the same volume as that of the cracking solution into the cracked mixed solution to blow, beat and uniformly mix;

(4) transferring all the liquid in the centrifuge tube to a new nuclease-free centrifuge tube by using a sterile gun head, adding 7-10 mu l of extraction magnetic beads and 5-10 mu l of nucleic acid settling agent into the mixed solution obtained in the step (3), covering a centrifuge tube cover, placing the centrifuge tube on a vertical mixing instrument, mixing for 10min at room temperature, and fully combining the magnetic beads with the genome DNA;

(5) taking down the centrifugal tube in the step (4), performing instantaneous centrifugation, placing the centrifugal tube on a magnetic frame for 2-3 min, and after the magnetic beads are completely adsorbed, absorbing and discarding the liquid,

(6) taking out the centrifuge tube from the magnetic frame, placing the centrifuge tube in the centrifuge tube plate (5), adding 200-400 μ l (preferably 200 μ l) of washing solution, shaking fully and mixing uniformly, centrifuging instantaneously, placing the centrifuge tube on the magnetic frame for 1min, sucking and removing the washing solution after the magnetic beads are completely adsorbed, and repeating the washing step; centrifuging the centrifugal tube instantaneously, placing the centrifugal tube on a magnetic frame, sucking residual liquid, and airing for 30 s;

(7) taking down the centrifugal tube in the step (6), adding 10-20 mul (preferably 15 mul) of eluent into the centrifugal tube, fully shaking and mixing uniformly, and then putting the centrifugal tube into a constant-temperature shaking and mixing instrument at 2000rpm, 55 ℃ and 10-20 min (preferably 10 min);

(8) and (4) taking out the centrifugal tube in the step (7) for instantaneous centrifugation, placing the centrifugal tube on a magnetic frame for 2-3 min, transferring the eluent into a new centrifugal tube for standby after the magnetic beads are completely adsorbed, or directly using the magnetic bead mixed solution added with the eluent in the step (7) for downstream PCR reaction or STR typing detection.

The invention has the beneficial effects that:

the nucleic acid precipitation aid used in the invention is acrylamide; when the nucleic acid is precipitated, 5-10 mul of the nucleic acid is added (when 5-10 mul of the nucleic acid precipitation aid is added into a 50-500ml system, the recovery rate of the nucleic acid can reach more than 90%), the yield of the nucleic acid precipitate can be obviously improved, the recovery rate of trace DNA can reach 98-100%, and meanwhile, short primer fragments and dNTP can be selectively removed.

The magnetic bead is silicon-based coated ferroferric oxide magnetic bead, and is coated by a polystyrene magnetic ring and silicon base; the magnetic beads are small in mass, slow in settling speed and long in suspension time when the magnetic field does not act, can be rapidly gathered at the speed 2 times of the magnetic responsiveness of the common magnetic beads under the action of the magnetic field, complete separation of the magnetic beads and a solution is instantly completed, especially the extraction rate of trace DNA is increased, the efficiency of extracting the DNA by the magnetic beads is improved, and meanwhile, sufficient carboxyl groups on the surfaces of the magnetic beads are combined with target DNA molecules in a sample to the maximum extent, especially the extraction rate of the trace DNA, so that the advantages are more obvious; when the Identifier Plus kit is used for STR typing detection, the mixed solution of the magnetic beads and the eluent can be directly loaded for PCR, the trace DNA loss is reduced, and the detection rate is improved.

Compared with the prior art that the detection rate of extracting the trace DNA is lower than 50%, the detection rate of extracting the trace DNA is higher than 75%. The invention creatively adopts a gradient cracking mode to effectively crack the DNA of the trace biological detection material, and simultaneously adds the nucleic acid precipitation aid for improving the recovery rate of the DNA, thereby obviously improving the extraction efficiency of the trace DNA, and further improving the detection rate, the accuracy and the sensitivity of the criminal investigation case evidence.

Meanwhile, the magnetic bead method adopted by the extraction can realize automatic extraction, realize parallel detection of multiple samples simultaneously, and save labor and time cost.

Drawings

FIG. 1 is a graph showing the result of electrophoretic verification of the amplification products of the internal control PCR system with the lowest sample loading in example 1. Reference numeral 1: PCR negative, 2: PCR positive, 3: 50ng stock solution, 4: 5pg, 5: 10pg, 6: 30pg, 7: 50pg, 8: 100pg, 9: 500pg, 10: 1000pg, 11: 2000pg, 12: 3000 pg.

FIG. 2 is a PCR amplification chart of a sample obtained by extracting a trace amount of cells in example 3.

FIG. 3 is a graph showing the results of the electrophoresis test of the PCR amplification products of trace DNA in example 4. Reference numeral 1: PCR negative, 2: PCR positive, 3: experiment blank control, 4 sign pens, 5: cell-phone screen, 6: cup, 7: table, 8: door handle, 9: wallet, 10: butts, 11: chewing gum, 12: underwear pieces, 13: cotton yarn gloves.

FIG. 4 is a graph showing the result of electrophoresis of the internal control PCR assay for the detection rate of DNA of single fingerprint-derived exfoliated cells in example 5. Reference numeral 1: negative control, 2: positive control, 3-42: and (4) sampling.

FIG. 5 shows the STR typing verification result of trace DNA detection rate. Wherein, 5-1: a sign pen; 5-2: a mobile phone screen; 5-3: a cup; 5-4: glass; 5-5: cigarette end, 5-6: chewing gum, 5-7: underwear fragments, 5-8: cotton yarn gloves.

FIG. 6 is a result of comparing the trace amount of DNA detection rate with that of the kit of the same type. Wherein 6-1 is an electrophoresis chart of an amplification product of DNA extracted by the kit and the extraction method, 1: a PCR negative control; 2: PCR positive control; 3-12: extracting products from a single fingerprint; 6-2 is commercialized D shield^TMElectrophoresis diagram of amplified product of DNA extracted by hypersensitive DNA extraction kit type II instruction extraction method, 1: a PCR negative control; 2: PCR positive control; 3-12: extracting the product from single fingerprint.

FIG. 7 is a diagram of STR typing detection results of 9 samples successfully amplified to obtain target bands in DNA extracted by the kit and the extraction method of the present invention. Wherein 7-1 to 7-9 correspond to 9 samples, respectively.

FIG. 8 is a commercial D shield^TMSTR typing detection result diagram of 4 samples successfully amplifying target bands in DNA extracted by type II extraction method of the hypersensitive DNA extraction kit. Wherein 8-1 to 8-4 correspond to 4 samples, respectively.

Detailed Description

To better illustrate the problems addressed by the present invention, the technical solutions adopted and the effects achieved, reference will now be made to the following detailed description and related information. It should be noted that the present disclosure includes, but is not limited to, the following examples and combinations thereof.

Example 1 internal control PCR System validation of minimum load of Trace DNA

An experiment is designed to verify an internal control PCR system with the lowest sample loading amount of trace DNA, and the lowest template amount capable of carrying out effective PCR reaction is determined.

The genomic DNA templates of 50 ng/. mu.l volunteers were subjected to gradient dilution to obtain total amounts of 3000pg, 2000pg, 1000pg, 500pg, 100pg, 50pg, 30pg, 10pg, and 5pg, respectively, and verified. To ensure the accuracy of the experiment, a control experiment was designed: the negative control sample loading template is 10mmol/L Tris, and is used for checking whether a reaction system is polluted by nucleic acid substances; the positive control template is Human genome DNA with the concentration of 50 ng/microliter, is purchased from Human genomicDNA with the concentration of century Kangkang, and is used for checking whether the amplification capacity of a reaction system is normal or not and whether a sample is abnormal or not; then 50ng of the total amount of the volunteer genome template stock solution and the positive template control are added.

The template, negative control and positive control of the gradient dilution are amplified according to the following internal control PCR system.

The internal control PCR amplification reaction system is (12.5 mu l system):

taq enzyme (containing dNTP 200. mu. mol/L, mg²⁺Solution 0.75mmol/L) 6.25. mu.l;

1 mul of amplification primer (0.4 mul/L) (amplification length 137 bp);

genomic DNA template 5.25. mu.l.

The above-mentioned amplification primer references "unknown response of differential unique genomic DNA sequences in the cell-free plasmid DNA of induced primers", ACTB1 primer sequence: GCGCCGTTCCGAAAGTT, CGGCGGATCGGCAAA, 137bp, unmethylated.

The internal control PCR reaction conditions are as follows:

after PCR amplification of all sample volumes is completed, 2. mu.l of PCR product is taken from each genomic DNA template dilution gradient, and is detected by 1%, 150V and 40min agarose gel electrophoresis, wherein the electrophoresis loading conditions are as follows: the PCR product was added in 2. mu.l + 3. mu. ltris: loading (Tris-HCl pH 8.0: loading buffer 2:1) to determine the lowest loading of genomic DNA under the PCR reaction. The results are shown in FIG. 1.

The detection result shows that the negative control has no non-specific strip except the residual primer, which indicates that the reaction system has no pollution; the positive control strip is bright and has correct size, which indicates that the amplification capacity of the reaction system is normal. The loading amount of DNA detected by the internal control PCR system is more than 50pg, and can successfully amplify 137bp target bands, while the loading amount of DNA detected by 30pg, 10pg and 5pg is not amplified to 137bp target bands, and the internal control PCR reaction system can detect that the lowest loading amount of genome DNA is 50 pg.

Example 2 validation of reagent component concentration versus detection Rate

Designing an orthogonal experiment, verifying the extraction rate of trace DNA by determining the NaCl concentration, the guanidinium isothiocyanate concentration and the sodium dodecyl sulfate concentration in the lysate, and designing a 3-factor-4 horizontal orthogonal experiment; 0.1mol/L, 0.5mol/L, 1mol/L and 2mol/L of Nacl; concentration of isothiocyanate: 2mol/L, 3mol/L, 4mol/L and 5 mol/L; concentration of SDS: 1%, 2%, 5%, 10%

In the experiment, 10^6 cultured cells/ml are used, diluted by 10 times, and about 100 cells in 1 microliter of cell suspension are taken for extraction

(1) Adding a suspension containing 100 cultured cells to a 1.5ml centrifuge tube;

(2) adding lysis solutions consisting of NaCl, guanidinium isothiocyanate and sodium dodecyl sulfate with different concentrations into the centrifugal tube with the template added in the step (1) according to an orthogonal experiment table, adding protease K, and blowing, beating and uniformly mixing;

(3) adding isopropanol into the solution obtained in the step (2), extracting magnetic beads and a nucleic acid precipitation aid, and mixing;

(7) instantaneously centrifuging the mixed solution obtained in the step (6), transferring the eluent into a new centrifugal tube, and carrying out concentration measurement

Experimental orthogonality table and results table:

table 1: experimental orthogonal table

Table 2: results analysis table

Factors of the fact	Sum of squares of deviation	Degree of freedom	F ratio	Critical value of F
					Concentration of NaCl	0.742	3	2.138	3.860
Guanidinium isothiocyanate concentration	0.252	3	0.726	3.860
					Concentration of SDS	0.047	3	0.135	3.860
Error of the measurement	1.04	9

The experimental result shows that the F value of NaCl is analyzed to be the largest, the influence of NaCl concentration on magnetic bead nucleic acid adsorption is prompted to be the largest, the prompt concentration is 0.5-1.0 mol/L, when guanidine thiocyanate is 2-4 mol/L and sodium dodecyl sulfate is in the range of 1% -5%, the total extraction amount is higher

Example 3 micro cell extraction validation

The experimental sample is a cultured lung cancer cell strain NCI-H1975, the concentration of the cultured cells is 10^ 6/ml, the cells are diluted by 100 times, about 10 cells of 1 mul cell suspension are taken for extraction,

(1) add 1. mu.l suspension containing 10 cultured cells to a 1.5ml centrifuge tube;

(2) adding 250 ul of lysis solution and 10 ul of proteinase k;

(3) fully cracking cells under the conditions of 2000rpm, 55 ℃ and 20min by utilizing the high-salt environment of the lysate, releasing cell genome DNA, and then adding isopropanol with the same volume as the lysate into the cracked mixed solution to blow, beat and uniformly mix;

(4) adding 7 mu l of extracted magnetic beads and 10 mu l of nucleic acid precipitation aid into the mixed solution obtained in the centrifugal tube, covering a centrifugal tube cover, placing the centrifugal tube on a vertical mixing machine, and mixing for 10min at room temperature to ensure that the magnetic beads are fully combined with the genome DNA;

(5) taking off the centrifugal tube, centrifuging instantaneously, placing the centrifugal tube on a magnetic frame for 3min, adsorbing the magnetic beads completely, removing the liquid,

(6) taking out the centrifuge tube from the magnetic frame, placing on a centrifuge tube plate, adding 200 μ l of washing solution, shaking thoroughly, mixing, centrifuging instantaneously, placing the centrifuge tube on the magnetic frame for 1min, adsorbing the magnetic beads completely, removing the washing solution, and repeating the washing step; centrifuging the centrifugal tube instantaneously, placing the centrifugal tube on a magnetic frame, sucking residual liquid, and airing for 30 s;

(7) taking off the centrifuge tube, adding preferably 15 μ l of eluent into the centrifuge tube, shaking and mixing, placing the centrifuge tube into a constant temperature shaking and mixing instrument at 2000rpm, 55 deg.C, 10min

(8) And taking out the centrifugal tube for instantaneous centrifugation, placing the centrifugal tube on a magnetic frame for 3min, transferring the eluent into a new centrifugal tube for standby after the magnetic beads are completely adsorbed, and performing downstream PCR verification.

The human EGFR gene mutation detection kit is adopted for carrying out fluorescence PCR detection, the CT value is less than 40, the detection success is realized, the marked- (minus) position is the result of being more than the CT value 40, and the detection results are shown in Table 3

Table 3: fluorescent PCR results

Sample numbering	Negative of	Positive for	1	2	3	4
							CT value	——	22.67	35.69	36.51	——	37.12
Sample numbering	5	6	7	8	9	10
							CT value	33.21	——	34.82	35.14	——	36.23

And (3) displaying a detection result: after the control of yin and yang was removed, 7 samples out of 10 samples could detect the EGFR T790M mutation site, which indicates that 7 samples out of 10 samples could be successfully extracted, and the detection results are shown in FIG. 2

Example 4 internal control PCR validation of Trace DNA detection Rate

Fingerprint and contact article types: sampling parts touched by the fingers of the volunteer, such as a sign pen, a mobile phone screen, a cup, a table, a door handle, a wallet and the like according to the description of the mucosa sampler,

other classes: such as cigarette butts, chewing gum, underwear pieces, cotton gloves, etc., a small sample (no more than 1cm x 1cm in size) is surgically cut.

The DNA extraction is carried out according to the kit and the extraction method, and the specific steps are as follows:

(1) fingerprint and contact article types: sticking the surface of the contact object by using a cast-off cell sticking device, and putting the side with viscosity after tearing off the mucous membrane into a nuclease-free 1.5ml centrifuge tube inwards;

other classes: a small sample is cut and put into a nuclease-free 1.5ml centrifuge tube;

adding 360 mul of lysis solution, and immersing the material to be tested; 10 μ l proteinase K (10mg/ml)

(2) Fully cracking a material evidence sample under the conditions of 2000rpm, 55 ℃ and 20min by utilizing the high-salt environment of a cracking solution to release the material evidence exfoliative cell genome DNA, and then adding isopropanol with the same volume as that of the cracking solution into the cracked mixed solution to blow, beat and uniformly mix;

(3) transferring all liquid in a 1.5ml centrifuge tube to a new nuclease-free 1.5ml centrifuge tube by using a sterile gun head, adding 7 mu l of extraction magnetic beads and 10 mu l of nucleic acid precipitation aid into the solution, covering a centrifuge tube cover, placing the centrifuge tube on a vertical mixer, mixing for 10min at room temperature, and fully combining the magnetic beads with the genomic DNA;

(4) taking off the centrifugal tube, centrifuging instantaneously, placing the centrifugal tube on a magnetic frame for 3min, adsorbing the magnetic beads completely, removing the liquid,

taking out the centrifuge tube from the magnetic frame, placing on a centrifuge tube plate, adding 200 μ l of washing solution, shaking thoroughly, mixing, centrifuging instantaneously, placing the centrifuge tube on the magnetic frame for 1min, adsorbing the magnetic beads completely, removing the washing solution, and repeating the washing step;

(5) centrifuging the centrifugal tube instantaneously, placing the centrifugal tube on a magnetic frame, sucking residual liquid, and airing for 30 s;

(6) taking down the centrifugal tube, adding 15 mul of eluent into the centrifugal tube, fully shaking and uniformly mixing, and then putting the centrifugal tube into a constant-temperature shaking and uniformly mixing instrument at 2000rpm, 55 ℃ and 10 min;

(7) and (3) taking out the centrifugal tube for instantaneous centrifugation, placing the centrifugal tube on a magnetic frame for 2min, transferring the eluent into a new centrifugal tube for standby after the magnetic beads are completely adsorbed, or directly using the magnetic bead mixed solution added with the eluent in the step (6) for downstream PCR reaction or STR typing detection.

After extraction, the internal control PCR reaction system in example 1 was used to perform PCR amplification on the above trace DNA, the loading of the PCR template was 5.25. mu.l, and positive and negative controls were set to ensure the accuracy of the experimental results. After the amplification is finished, 2 μ l of each PCR product is detected by agarose gel electrophoresis at 1%, 150V and 40min, and the conditions of electrophoresis loading are as follows: PCR product 2. mu.l + 3. mu.l Tris loading (Tris-HCl pH8.0 loading buffer 2:1) was counted for detection rate. The results are shown in FIG. 3.

The detection result shows that the negative control has no non-specific strip except the residual primer, which indicates that the reaction system has no pollution; the positive control strip is bright and has correct size, which indicates that the amplification capacity of the reaction system is normal. Target strips are successfully amplified in the detection results of the trace biological sample, and the success rate of DNA extraction is 100%.

Example 5 internal control PCR verification of DNA detection rate of single fingerprint exfoliated cell

The surfaces of articles such as sign pens, mobile phone screens, cups, glass, tables, door handles, mice, keyboards, doorbells, card readers and the like which are touched by fingers frequently are cleaned by ultrapure water, the articles are wiped to be dry by using dust-free paper, the surfaces of the treated articles are pressed by a single finger for about 30-60 seconds, 40 samples of volunteers are collected according to the method, and the samples are sampled according to the description of a mucosa sampler. All samples were subjected to DNA extraction according to the kit and extraction method of the present invention in example 2. After extraction, the single fingerprint exfoliated cell DNA is subjected to PCR amplification by using the internal control PCR reaction system in the embodiment 1, the sample loading amount of a PCR template is 5.25 mu l, and the accuracy of an experimental result is ensured by setting a negative control and a positive control. After the amplification is finished, 2 mul of PCR products are respectively detected by agarose gel electrophoresis at 1%, 150V and 40min, and the conditions of electrophoresis loading are as follows: PCR product 2. mu.l + 3. mu.l Tris loading (Tris-HClpH 8.0 loading buffer 2:1) was counted for detection rate. The results are shown in FIG. 4.

The detection result shows that the negative control has no non-specific strip except the residual primer, which indicates that the reaction system has no pollution; the positive control strip is bright and has correct size, which indicates that the amplification capacity of the reaction system is normal. 31 samples in 40 single fingerprint cast-off cell DNA samples successfully expand target strips, and the detection rate of the single fingerprint cast-off cell DNA reaches 77.5 percent.

Example 6 STR typing verification of trace DNA detection Rate

DNA extraction was performed on a sign pen, a mobile phone screen, a cup, a table touched by the hand of the volunteer, and a cigarette butt, chewing gum, underwear pieces, cotton gloves used by the volunteer according to the kit and the extraction method of the present invention in example 2. The downstream mode of forensic detection of trace DNA is usually STR typing for identification. After the extraction is finished, mixed liquor containing magnetic beads or eluent without the magnetic beads can be selected as a DNA template, an Identifier Plus kit of American ABI company is selected for amplification, a 3500xl fluorescence analyzer is used for capillary electrophoresis, Gene Mapper software is used for allele analysis, and the detection rate is counted. The results are shown in FIG. 5.

The detection result shows that the DNA obtained by the trace organism test material according to the kit and the extraction method of the invention is subjected to STR typing detection, the result is that 8 samples can detect 12 or more loci, and the result is consistent with the DNA typing result of volunteers, and the detection result is shown in figure 5.

Example 7 verification of Trace DNA detection Rate versus kit of the same product

The sampling method comprises the steps of sampling a sign pen, a mobile phone screen, a cup, a table, a mouse and a keyboard touched by hands of a volunteer, and cigarette butts, chewing gums, underwear fragments and cotton gloves used by the volunteer respectively, parallelly collecting two samples for each micro-contact biological detection material, wherein the samples are 20 samples in total, and averagely dividing the 20 samples into 2 samples and 10 samples according to the difference of the micro-contact biological detection materials. DNA extraction was performed on 10 scene physical evidence in one part according to the kit and the extraction method of the invention in example 2; the other 10 scene material evidences are according to a commercialized D shield^TMDNA extraction is carried out by the hypersensitive DNA extraction kit II type instruction extraction method. After extraction, the internal control PCR reaction system in example 1 was used to perform PCR amplification on the above trace DNA, the loading of the PCR template was 5.25. mu.l, and positive and negative controls were set to ensure the accuracy of the experimental results. After the amplification is finished, 2 mul of PCR products are respectively detected by agarose gel electrophoresis at 1%, 150V and 40min, and the conditions of electrophoresis loading are as follows: PCR product 2. mu.l + 3. mu.l Tris loading (Tris-HCl pH8.0 loading buffer 2:1) was counted for detection rate. The detection result is shown in FIG. 6, wherein 6-1 is an electrophoresis chart of the amplification product of the DNA extracted by the kit and the extraction method of the invention; 6-2 is commercialized D shield^TMElectrophoresis chart of amplified product of DNA extracted by hypersensitive DNA extraction kit type II instruction extraction method.

The detection result shows that the negative control has no non-specific band except the residual primer, which indicates the reactantIs pollution-free; the positive control strip is bright and has correct size, which indicates that the amplification capacity of the reaction system is normal. The kit and the extraction method of the invention can successfully expand target bands from 9 samples out of 10 samples, the detection rate is 90 percent, and the D shield^TMType II hypersensitive DNA extraction kit for extracting 4 successfully amplified target bands from 10 specimens, with detection rate of 40%

And simultaneously, samples successfully amplified to obtain target bands in the two batches of samples are taken to carry out STR typing detection, DNA extraction is carried out by the kit and the extraction method, and the result shows that 9 samples successfully amplified to obtain the target bands can detect 12 or more sites and is consistent with the DNA typing result of volunteers (the DNA typing of the volunteers is shown in a table 3), and the detection result is shown in a table 7.

Commercialized D shield^TMDNA extraction is carried out by a type II extraction method of the hypersensitive DNA extraction kit, and 12 or more sites can be detected by 4 successful target band amplification results, as shown in figure 8.

Table 3: volunteer DNA typing table

STR locus	D8S1179	D21S11	D7S820	CSF1PO	D3S1358
						Typing
	9/15	29/31	12	10/11	12/14
						STR locus	TH01	D13S317	D16S539	D2S1338	D19S433
Typing
		6/11	8/10	10/14	20/22	10/11
STR locus							VWA	TPOX	D18S51	D5S818	FGA
	Typing	11/19	8/12	14/25	11	18/30

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims

1. A kit for extracting trace DNA, the reagent comprising: extracting magnetic beads, lysis solution, protease K solution, washing solution, nucleic acid precipitation aid and eluent from DNA;

the DNA extraction magnetic beads are silicon-based coated ferroferric oxide magnetic beads, and the structure from inside to outside is as follows: the magnetic microsphere core, the polystyrene sealing magnet ring and the silicon dioxide coating functional group layer;

the lysis solution comprises: guanidine isothiocyanate with the final concentration of 2-5 mol/L, Tris-HCl with the final concentration of 50-100 mmol/L, sodium dodecyl sulfate or sodium dodecyl sarcosinate with the final concentration of 1-10% and EDTA with the final concentration of 1-10 mmol/L, Trition X-100 with the volume percentage concentration of 0.5-7% and soluble salt NaCl with the final concentration of 0.1-1 mol/L;

the washing liquid comprises: Tris-HCL with the final concentration of 5-20mmol/L and ethanol with the volume percentage concentration of 70-80%;

the concentration of the protease K is 5-10 mg/ml;

the nucleic acid precipitation aid is 1.5% of acrylamide;

the eluent comprises: Tris-HCl at a final concentration of 5-20mmol/L, pH 8.0.

2. The kit of claim 1, wherein the magnetic beads have a diameter of 200 to 2000nm and a functional group density of 1.0 to 5 μ M/mg.

3. The kit according to claim 1, wherein the concentration of the proteinase K solution is 10 mg/ml.

4. The kit of claim 1, wherein the lysis solution comprises: guanidine isothiocyanate with the final concentration of 3mol/L, Tris-HCl with the final concentration of 50mmol/L, sodium dodecyl sulfate with the final concentration of 2 percent, EDTA with the final concentration of 10mmol/L, Trition X-100 with the volume percentage of 2 percent and soluble salt NaCl with the final concentration of 1 mol/L.

5. The kit of claim 1, wherein the wash solution comprises: Tris-HCL with the final concentration of 10mmol/L and ethanol with the volume percentage concentration of 80 percent.

6. The kit of claim 1, wherein the elution fluid comprises: Tris-HCl at a final concentration of 10mmol/L, pH 8.0.

7. A method for extracting trace DNA by using the kit of any one of claims 1 to 6, comprising the steps of:

(1) lysing the sample cells;

(7) and (4) instantly centrifuging the mixed solution obtained in the step (6), transferring the eluent into a new centrifugal tube for standby, or directly using the mixed solution of the magnetic beads with the eluent added in the step (6) for downstream detection.

8. The method according to claim 7, characterized by the following specific steps:

(1) putting a sample into a centrifuge tube, adding 1-10 mul of proteinase K, and adding 250-500 mul of lysis solution in volume;

(2) fully cracking the material evidence sample by the mixed solution in the step (1) under the conditions of 2000rpm, 55 ℃ and 10-20 min, and then adding isopropanol with the same volume as that of the cracking solution into the cracked mixed solution to be uniformly mixed;

(3) adding 7-10 mul of extraction magnetic beads and 5-10 mul of nucleic acid precipitation aid into the mixed solution obtained in the step (2), and mixing for 10 min;

(4) instantly centrifuging the mixed solution obtained in the step (3), placing the centrifugal tube on a magnetic frame for 2-3 min, and sucking away the liquid after the magnetic beads are completely adsorbed;

(5) adding 200-400 mu l of washing solution into the centrifuge tube in the step (4), shaking fully and mixing uniformly, then centrifuging instantly, placing the centrifuge tube on a magnetic frame for 1min, absorbing the washing solution after the magnetic beads are completely adsorbed, repeating the washing step once, and airing for 30 s;

(6) adding 10-20 mul of eluent into the centrifugal tube in the step (5), fully shaking and mixing uniformly, and then putting the centrifugal tube into a constant-temperature shaking and mixing instrument at 2000rpm, 55 ℃ and 10-20 min;

(7) and (4) instantly centrifuging the mixed solution obtained in the step (6), placing the centrifugal tube on a magnetic frame for 2-3 min, and transferring the eluent into a new centrifugal tube for standby after the magnetic beads are completely adsorbed, or directly using the magnetic bead mixed solution added with the eluent in the step (6) for downstream detection.