Multiple SNP combined micro haplotype amplification system based on one-step library establishment, construction method, product and application

Abstract

The invention belongs to the technical field of forensic medicine, and particularly relates to an amplification system of multiple SNP combined micro-haplotypes based on one-step library construction, a construction method, a product and application. According to the present invention, based on the RC-PCR technology, the one-step library building composite amplification system is constructed, and the amplification system comprises an RC-Primer represented by the sequence represented by SEQ ID No.1-SEQ ID No.210, an I5-Primer represented by the sequence represented by SEQ ID No.211-SEQ ID No.214, and an I7-Primer represented by the sequence represented by SEQ ID No.215-SEQ ID No.222; according to the method, one-time PCR reaction is achieved, targeted amplification and library construction are completed at the same time, the experiment time is greatly shortened, the experiment steps are simplified, and the advantages are more remarkable when a large number of samples are detected at the same time.

Description

Technical Field

[0001] The present invention belongs to the field of forensic medicine technology, and specifically relates to a multiple SNP combined with micro-haplotype amplification system based on one-step library construction, as well as a construction method, product and application. Background Art

[0002] Next-generation sequencing (NGS), also known as high-throughput sequencing, is widely used in forensic genetics due to its high throughput, rapid sequencing speed, high accuracy, and low cost. Based on the principles of NGS, before sequencing a DNA sample, sequencing primers are added to both ends of the DNA to initiate the sequencing reaction, a tag sequence to distinguish samples, and an anchor sequence that connects to the sequencing chip. This step is called library construction. This step differs from conventional first-generation sequencing and is cumbersome, requiring a lengthy experimental process and high laboratory skills.

[0003] Currently, the most common library construction method is TA cloning, which involves the following steps: target site amplification, end repair, A-tailing, and T-tailed adapter ligation, interspersed with magnetic bead purification or magnetic bead sorting. This method is relatively mature, but due to its numerous steps and lengthy experimental workflow, it requires high levels of laboratory expertise, making it particularly time-consuming and labor-intensive when constructing libraries for large numbers of samples.

[0004] With the development of library construction technology, the more commonly used library construction method for targeted sequencing is the two-step library construction method. Its main method is: the ends of the amplification primers for the first step of targeted amplification are designed to add specific sequences that can complement the amplification primers in the second step. After the first step of PCR, a targeted product with complementary pairing with the amplification primers in the second step is obtained. Then, the sequencing adapter is connected through the second step of PCR reaction to obtain a library that can be used for sequencing on the machine. Magnetic bead purification is required after each amplification. From the start of amplification to the completion of magnetic bead purification, the total experimental time of this method is about 5.5-7.5 hours (the total time consumed varies slightly for different amplification systems). In addition, there is a risk of contamination of PCR products due to multiple transfers.

[0005] Therefore, there is an urgent need to develop a method that is simple to operate, takes less time, and has higher detection efficiency and accuracy. Summary of the Invention

[0006] This paper, based on Reverse Complement PCR (RC-PCR) technology, designs a one-step library construction method. This method simplifies the experimental steps, requiring only a single PCR step and a single magnetic bead purification step to complete library construction, and the PCR time is only 1 hour and 42 minutes. This significantly shortens the experimental time, reduces the number of steps, and reduces the risk of contamination, helping to improve the efficiency and accuracy of forensic identification.

[0007] The technical solution of the present invention is:

[0008] On the one hand, the present invention provides a multiple SNP combined with micro-haplotype amplification system based on one-step library construction, the amplification system comprising a primer combination for simultaneously amplifying 80 high MAF value SNP sites, 1 Y-Indel and 24 micro-haplotype sites;

[0009] The 80 SNP sites with high MAF values include rs1253805, rs565645, rs11208133, rs681968, rs1171043, rs880329, rs7515856, and rs891700 on chromosome 1; rs876724, rs950661, rs12622958, rs6754768, rs1385172, rs6705667, rs7597826, rs6714809, and rs6436688 on chromosome 3; rs6793085, rs7649122, rs9310740, and rs57579284 on chromosome 4. rs2295499, rs3096579, rs2048961, rs10034991, rs7668855, rs1605463, rs6535598, rs10034372 on chromosome 4, rs10866562, rs252369, rs10472761, rs272735, rs61467430, rs264748, rs13182883 on chromosome 5, rs3799000, rs2747740, rs214955, rs316032, rs9366013 on chromosome 6, rs2686920 on chromosome 7, rs13182883 on chromosome 8 rs10092491, rs7815202 on chromosome 9, rs10758556, rs7046317, rs10821241, rs1463729 on chromosome 10, rs1418407, rs10826551, rs7088769, rs11146518 on chromosome 11, rs1319309 on chromosome 11, rs2269355, rs6581429, rs959897 on chromosome 12, rs9510177, rs6563522, rs12585235, rs1602204, rs354439 on chromosome 14 958395, rs1454361, rs7157655, rs1956467, rs1202668, rs11846252, rs7151788, rs8041340 on chromosome 15, rs8050385, rs9928480, rs8043925 on chromosome 16, rs9951171, rs28690792, rs3991209 on chromosome 18, rs576261 on chromosome 19, rs6041563 on chromosome 20, rs2070650 on chromosome 21, rs361844, rs1028528 on chromosome 22;

[0010] The 1 Y-Indel consists of rs34733631;

[0011] The 24 microhaplotype loci consist of F1-012, F3-019, F6-026, F7-0210, F9-033, F12-045, F16-057, F17-058, F18-061, F22-071, F23-072, F24-074, F26-086, F27-092, F28-101, F29-107, F30-114, F31-117, F32-122, F33-124, F34-131, F36-164, F38-175, and F39-184;

[0012] The primer combination includes RC-Primer, I5-Primer and I7-Primer, and the design method thereof is as follows:

[0013] S1. Design conventional primers based on the target sequence;

[0014] S2. Design reverse complementary primer RC-Prime based on conventional primers;

[0015] S3. Design the I5 / I7 end adapter primers I5-Primer and I7-Primer;

[0016] The RC-Primer includes a reverse complementary sequence to the complementary regions of I5-Primer and I7-Primer;

[0017] The 3' end of the RC-Primer is blocked with TAGGTGT, and the last GT base is thiolated and the last base T is phosphorylated.

[0018] Specifically, the sequences of the RC-Primers are shown as SEQ ID No. 1 to SEQ ID No. 210;

[0019] The sequences of the I5-Primers are shown in SEQ ID No. 211 to SEQ ID No. 214;

[0020] The sequences of the I7-Primer are shown as SEQ ID No. 215 to SEQ ID No. 222.

[0021] Specifically, the amount of RC-Primer added was 0.25 μM, and the amounts of I5-Primer and I7-Primer added were both 10 μM.

[0022] Specifically, the one-step library construction and amplification system further includes one or more of a buffer, dNTPs, and a high-fidelity enzyme.

[0023] In another aspect, the present invention provides a library construction method based on multiple SNPs and micro-haplotypes, wherein the library construction method comprises performing PCR amplification on the sample using the aforementioned amplification system.

[0024] Specifically, the PCR amplification procedure is shown in the following table:

[0025]

[0026] In the PCR amplification program, 68-60°C means that the temperature is decreased by 0.5°C in each cycle after the first cycle at 68°C.

[0027] Specifically, the sample includes one or more of blood, blood spots, oral cells, semen, semen stains, bones, hair, saliva, saliva stains, sweat, amniotic fluid containing fetal cells, or urinary tract exfoliated cells.

[0028] In another aspect, the present invention provides a kit comprising the aforementioned amplification system.

[0029] Specifically, the kit also includes a library purification kit.

[0030] Preferably, the library purification kit comprises one or more of a purification column or magnetic beads, a binding buffer, a washing buffer and an elution buffer.

[0031] In another aspect, the present invention provides the use of the aforementioned amplification system or library construction method or kit in forensic rapid individual identification.

[0032] The beneficial effects of the present invention are:

[0033] (1) The typing results of library construction based on the one-step library construction method of the present invention have good reproducibility.

[0034] (2) The library construction based on the one-step library construction method of the present invention has a certain ability to resist humic acid, EDTA, and indigo inhibition.

[0035] (3) The amplification system established based on the one-step library construction method of the present invention has good species specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the core principle of one-step library construction.

[0037] Figure 2 This is a comparison of 3' end blocking methods. DETAILED DESCRIPTION

[0038] The present invention will be further clarified and fully described below by way of examples. The following examples are only a portion of the present invention and are not intended to limit the present invention, but are merely for illustration. The experimental methods used in the following examples are all routine experiments unless otherwise specified, and the materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0039] Example 1 One-step library construction method

[0040] The core principle of the one-step library construction method provided by the present invention is as follows Figure 1 As shown, to construct a one-step library construction and targeted amplification system, first Figure 1 As shown in ①, design conventional PCR amplification primers; then design conventional PCR amplification primers according to Figure 1 The RC-Primer at ② in the figure contains the reverse complementary sequence of the amplification primer and can be used with Figure 1 The reverse complementary sequence of the I5-Primer / I7-Primer complementary region at ③ in the figure, and a blocking sequence to stop the amplification reaction.

[0041] RC-Primer and I5-Primer / I7-Primer are put into the PCR reaction system. At the beginning of the PCR reaction, RC-Primer can combine with I5-Primer / I7-Primer to generate Figure 1 The Final-Primer at position ④ in the figure contains the sequencing anchor sequence, barcode, sequencing primer, and targeting primer. Finally, the Final-Primer binds to the DNA template to generate the sequencing library, completing library construction.

[0042] The specific contents are as follows:

[0043] (1) RC-Primer 3' end blocking method: Use the non-human genomic DNA sequence "TAGGTGT" for blocking, perform thiolation modification between the last "GT" bases, and perform phosphorylation modification on the last base "T". The complete blocking sequence is: "TAGGTG*TP".

[0044] (2) One-step library construction PCR reaction system: The total system volume is 30 μL, the recommended starting DNA input is 3.5-20 ng, and the enzyme is produced by Yisheng Company, Cat: 10154ES03. The reaction system is shown in Table 1:

[0045] Table 1

[0046] Components Volume (μL) ddH2O 7-X 2×Hieff Canace 15 I5-Primer (10 μM) 2 I7-Primer (10 μM) 2 RC-Primer MIX (0.25 μM) 3 gDNA X

[0047] Note: X in the table indicates the gDNA input volume.

[0048] (3) One-step library construction PCR reaction procedure is shown in Table 2:

[0049] Table 2

[0050]

[0051]

[0052] Note: 68-60℃ means the temperature drops by 0.5℃ after the first cycle at 68℃.

[0053] (4) Magnetic bead purification

[0054] a. Take Beckman Agencourt AM Pure XP magnetic beads (Beckman Coulter, USA) out of the 4°C refrigerator and equilibrate at room temperature for 30 minutes.

[0055] b. Transfer 30 μL of PCR product to a 1.5 mL EP tube and add 54 μL of BeckMan magnetic beads (1.8 times the volume). Mix by pipetting or vortexing and incubate at room temperature for 5 minutes.

[0056] c. After incubation, place the EP tube on a magnetic stand for 3 minutes to allow the solution to clarify.

[0057] d. Completely remove the supernatant, add 180 μL of 70% ethanol solution to the EP tube, rotate the EP tube 180°, let it rest for 10 seconds, and rotate the EP tube 180° again.

[0058] e. Completely remove the supernatant, add 190 μL of 70% ethanol solution to the EP tube, rotate the EP tube 180°, let it rest for 10 seconds, and rotate the EP tube 180° again.

[0059] f. Cover the tube cap and centrifuge briefly to remove the remaining ethanol to the bottom of the tube. Place the EP tube on a magnetic stand and carefully use a 10μL pipette to remove the remaining ethanol at the bottom, taking care not to absorb the magnetic beads.

[0060] g. Place the EP tube on the magnetic rack and let it sit at room temperature for 3-5 minutes until the magnetic beads are dry and the residual ethanol is completely evaporated.

[0061] h. Add 24 μL DNase / RNase-Free Water (Thermo Scientific, USA), remove the EP tube from the magnetic stand, pipette or vortex to mix, and let it stand at room temperature for 5 minutes.

[0062] i. After instant centrifugation, place the EP tube on a magnetic rack for 2 minutes to allow the solution to clarify.

[0063] j. Use a pipette to aspirate 19 μL of supernatant and transfer it to a new EP tube containing the magnetic bead-purified library sample. Mark the tube.

[0064] (5) Comparison of 3' end blocking methods in comparative examples

[0065] According to the reaction system, reaction primers and reaction procedures of Example 1, the RC-primer 3' end blocking method was changed to compare the effects of various 3' end blocking methods on this method. The blocking methods are shown in Table 3:

[0066] Table 3

[0067]

[0068]

[0069] Note: “*” indicates thiolation modification; “-P” indicates phosphorylation modification.

[0070] The results are as follows Figure 2 (the target fragment is between 300-400 bp). It can be seen that the 3' end blocking method adopted by the present invention is most conducive to the one-step library construction method established by the present invention.

[0071] Example 2 Construction of a forensic SNP combined with microhaplotype multiplex system based on a one-step library construction method

[0072] Based on the method of Example 1, a one-step library construction multiplex SNP + micro-haplotype MH complex amplification system was constructed for routine forensic investigation. The multiplex system contains 80 high MAF value SNP sites, 1 Y-Indel and 24 micro-haplotype sites. The individual recognition rate of the system reaches 1-2.0265×10 -39 This study provides a new method for rapid individual identification using forensic next-generation sequencing. This study strictly adhered to the ethical guidelines of the Declaration of Helsinki. Blood samples were collected from 74 volunteers via venous sampling and stored in EDTA-anticoagulant tubes.

[0073] 2.1 SNP site selection results

[0074] Based on the autosomal SNP locus information in the 1000Genomes Project and Genome Aggregation Database, SNP loci were screened using the following criteria:

[0075] ① Screening out SNP sites with MAF>0.4 in the global population;

[0076] ② Simultaneously select SNP sites with MAF>0.4 in East Asian populations;

[0077] ③ Autosomal SNP sites added to the Verogen ForenSeq DNA Signature Prep Kit from Illumina, the Precision ID Identity Panel from Thermo Fisher, and the Forensic SNP Detection System from BGI;

[0078] ④ Simultaneously delete SNP sites within 1Mb of each other (to ensure linkage equilibrium between sites);

[0079] ⑤ According to the different lengths of each chromosome, the number of SNPs on each chromosome is allocated (to avoid the situation where the sites are concentrated on only a few chromosomes);

[0080] ⑥ Delete sites where there are other mutations upstream and downstream, making primer design difficult.

[0081] The selection results are shown in Table 4:

[0082] Table 4

[0083]

[0084]

[0085]

[0086] Note: Ref is wild type typing; Allele% is allele frequency; Alt is mutant typing; MAF is minimum allele frequency; del is deletion.

[0087] 2.2 Results of microhaplotype site selection

[0088] This study strictly adhered to the ethical guidelines of the Declaration of Helsinki. Blood samples were collected from 95 volunteers by venous blood sampling and stored in EDTA anticoagulant tubes.

[0089] To compensate for the shortcomings of SNP loci in mixed typing, this study further screened MH loci and incorporated them into the multiplex amplification system based on the one-step library construction method. The screening conditions were as follows:

[0090] ①Sequences with fragments within 230 bp and containing two or more (as many as possible) SNPs with MAF>0.01 were selected as microhaplotype sites;

[0091] ② Delete sites where there are mutations upstream and downstream that make primer design difficult;

[0092] ③ In addition, to ensure linkage equilibrium, sites with a distance of less than 1 Mb were deleted (MH sites with a distance of less than 1 Mb from the finally screened SNP sites were also not selected).

[0093] The selection results are shown in Table 5 and Table 6:

[0094] Table 5

[0095]

[0096]

[0097] Note: Ae value is the effective allele number. The larger the Ae value, the better the polymorphism of the MH locus. DP value is the individual recognition ability. The higher the DP value, the higher the individual recognition efficiency of the MH locus. The maximum value is 1.

[0098] Table 6

[0099]

[0100]

[0101]

[0102]

[0103]

[0104] Note: POS is the position of the SNP site in the reference genome hg38; MH is the abbreviation for microhaplotype; Rs is the rs number of the SNP site; SNP is the abbreviation for single nucleotide polymorphism; Ref is the wild-type typing; Alt is the mutant typing; Data from is the data source database; gnomAD is the Genome Aggregation Database; ALFA is the Allele Frequency Aggregator; 1KG is the 1000 Genomes Project; 1KG 30× is the 1000 Genomes Project 30×.

[0105] 2.3 Requirements for conventional amplification primer design

[0106] ① Primer length is between 16-35bp;

[0107] ②The length of the amplified fragment is between 81-116 bp;

[0108] ③ The Tm values of primers at each locus should be as consistent as possible;

[0109] ④ Avoid the formation of dimers and hairpin structures between primers;

[0110] ⑤ Try to avoid the presence of multiple consecutive identical bases in the primer; ⑥ The amplification product obtained by the primer in the target range is single.

[0111] 2.4 One-step library construction primer design results

[0112] 2.4.1 RC-Primer Design Results

[0113] The results are shown in Table 7:

[0114] Table 7

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123] Note: “*” indicates thiolation modification; “-P” indicates phosphorylation modification. 2.4.2 Illumina I5 / I7-Primer design results

[0124] The results are shown in Table 8:

[0125] Table 8

[0126]

[0127] Note: The underlined part is the barcode.

[0128] Example 3: Repeatability verification results of the forensic SNP combined with microhaplotype multiplex system constructed based on the one-step library construction method

[0129] According to the reaction system, reaction primers, and reaction procedure described in Example 1, a composite one-step library was constructed using the 9947A DNA standard and 3.5 ng of DNA from a male Han Chinese from northern China. Next-generation sequencing was performed on an Illumina platform Novasek X Plus sequencer. Gender loci were accurately typed across the three sequencing runs, with 100% accuracy for each locus across the three replicates. The results are shown in the table below, demonstrating the excellent reproducibility of the typing results using the one-step library construction method. This study strictly adhered to the ethical guidelines of the Declaration of Helsinki. A blood sample was collected from a male volunteer by venous blood sampling and stored in an EDTA anticoagulant tube.

[0130] The results are shown in Table 9, Table 10 and Table 11:

[0131] Table 9

[0132]

[0133]

[0134]

[0135] Table 10

[0136]

[0137]

[0138] Table 11

[0139]

[0140]

[0141] The results in Tables 9, 10 and 11 show that according to the experimental results, the system can maintain good result repeatability when the input amount of DNA is 3.5 ng.

[0142] Example 4 Results of the Forensic SNP Combined Microhaplotype Multiplex Anti-Inhibitor System Based on the One-Step Library Construction Method

[0143] According to the reaction system, reaction primers, and reaction procedure of Example 3, humic acid, EDTA, and indigo were added to the PCR amplification system to final concentrations of 25 μmol and 50 μmol, respectively. 10 ng of DNA from a Han Chinese female from northern China was added to the humic acid inhibition system. 10 ng of DNA from a Han Chinese male from northern China was added to the EDTA and indigo inhibition system. Next-generation sequencing was performed on the Illumina platform Novasek X Plus sequencer. The results showed that typing accuracy reached 100% at two concentrations of different inhibitors, indicating that library construction based on the one-step library construction method has a certain degree of resistance to inhibition by humic acid, EDTA, and indigo. This study strictly adhered to the ethical guidelines of the Declaration of Helsinki. Blood samples were collected from two volunteers by venous blood sampling and stored in EDTA anticoagulant tubes.

[0144] The results are shown in Table 12, Table 13 and Table 14:

[0145] Table 12

[0146]

[0147]

[0148]

[0149] Table 13

[0150]

[0151]

[0152] Table 14

[0153]

[0154]

[0155] The results in Tables 12, 13 and 14 show that accurate analysis results can be obtained at 25 μmol / L and 50 μmol / L concentrations of humic acid, EDTA and indigo.

[0156] Example 5: Species-specific detection results of a forensic SNP combined with microhaplotype multiplex system based on a one-step library construction method

[0157] According to the reaction system, reaction primers, and reaction procedure of Example 1, 10 ng of DNA from chicken, cattle, pig, mouse, rabbit, and sheep was used to construct a composite system one-step library. Next-generation sequencing was performed on an Illumina platform Novasek X Plus sequencer. The results are shown in Table 15:

[0158] Table 15

[0159] chicken ox sheep pig mouse rabbit Number of SNP sites detected 0 1 2 0 2 0 Number of microhaplotype loci (MH) detected 1 1 1 1 0 0

[0160] As shown in Table 15, the amplification system established based on the one-step library construction method of the present invention has good species specificity, and non-human DNA cannot be successfully constructed and sequenced.

[0161] The above detailed description is a specific description of one feasible embodiment of the present invention, and this embodiment is not intended to limit the scope of the present invention. It should be noted that any equivalent implementation or modification that does not depart from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the scope of protection of the present invention shall be based on the attached claims.

Claims

1. A multiple SNP combined with micro-haplotype amplification system based on one-step library construction, characterized in that: The one-step library construction and amplification system includes a primer combination for simultaneously amplifying 80 high MAF value SNP sites, 1 Y-Indel and 24 minor haplotype sites; The 80 SNP sites with high MAF values include rs1253805, rs565645, rs11208133, rs681968, rs1171043, rs880329, rs7515856, and rs891700 on chromosome 1; rs876724, rs950661, rs12622958, rs6754768, rs1385172, rs6705667, rs7597826, rs6714809, and rs6436688 on chromosome 3; rs6793085, rs7649122, rs9310740, and rs57579284 on chromosome 4. rs2295499, rs3096579, rs2048961, rs10034991, rs7668855, rs1605463, rs6535598, rs10034372 on chromosome 4, rs10866562, rs252369, rs10472761, rs272735, rs61467430, rs264748, rs13182883 on chromosome 5, rs3799000, rs2747740, rs214955, rs316032, rs9366013 on chromosome 6, rs2686920 on chromosome 7, rs13182883 on chromosome 8 rs10092491, rs7815202 on chromosome 9, rs10758556, rs7046317, rs10821241, rs1463729 on chromosome 10, rs1418407, rs10826551, rs7088769, rs11146518 on chromosome 11, rs1319309 on chromosome 11, rs2269355, rs6581429, rs959897 on chromosome 12, rs9510177, rs6563522, rs12585235, rs1602204, rs354439 on chromosome 14 958395, rs1454361, rs7157655, rs1956467, rs1202668, rs11846252, rs7151788, rs8041340 on chromosome 15, rs8050385, rs9928480, rs8043925 on chromosome 16, rs9951171, rs28690792, rs3991209 on chromosome 18, rs576261 on chromosome 19, rs6041563 on chromosome 20, rs2070650 on chromosome 21, rs361844, rs1028528 on chromosome 22; The 1 Y-Indel consists of rs34733631; The 24 microhaplotype loci consist of F1-012, F3-019, F6-026, F7-0210, F9-033, F12-045, F16-057, F17-058, F18-061, F22-071, F23-072, F24-074, F26-086, F27-092, F28-101, F29-107, F30-114, F31-117, F32-122, F33-124, F34-131, F36-164, F38-175, and F39-184; The primer combination includes RC-Primer, I5-Primer and I7-Primer, and the design method thereof is as follows: S1. Design conventional primers based on the target sequence; S2. Design reverse complementary primer RC-Prime based on conventional primers; S3. Design the I5 / I7 end adapter primers I5-Primer and I7-Primer; The RC-Primer includes a reverse complementary sequence to the complementary regions of I5-Primer and I7-Primer; The 3' end of the RC-Primer is blocked with TAGGTGT, and a thiolation modification is performed between the last GT bases, and the last base T is phosphorylated.

2. The amplification system according to claim 1, characterized in that The sequences of the RC-Primers are shown in SEQ ID No. 1 to SEQ ID No. 210; The sequences of the I5-Primers are shown in SEQ ID No. 211 to SEQ ID No. 214; The sequences of the I7-Primer are shown as SEQ ID No. 215 to SEQ ID No.

222.

3. The amplification system according to claim 2, characterized in that The amount of RC-Primer added was 0.25 μM, and the amounts of I5-Primer and I7-Primer added were both 10 μM.

4. The amplification system according to any one of claims 1 to 3, characterized in that The one-step library construction and amplification system further comprises one or more of a buffer, dNTPs and a high-fidelity enzyme.

5. A library construction method based on multiple SNPs and micro-haplotypes, characterized in that: The library construction method comprises performing PCR amplification on the sample using the amplification system according to any one of claims 1 to 4.

6. The library construction method according to claim 5, characterized in that: The procedure of the PCR amplification is shown in the table below: In the PCR amplification program, 68-60°C means that the temperature is decreased by 0.5°C in each cycle after the first cycle at 68°C.

7. The library construction method according to claim 5 or 6, characterized in that: The sample includes one or more of blood, blood spots, buccal cells, semen, semen stains, bones, hair, saliva, saliva stains, sweat, amniotic fluid containing fetal cells, or urinary tract exfoliated cells.

8. A kit, characterized in that The kit comprises the amplification system according to any one of claims 1 to 4.

9. The kit according to claim 8, characterized in that The kit also includes a library purification kit.

10. Use of the amplification system according to any one of claims 1 to 4, the library construction method according to any one of claims 5 to 7, or the kit according to any one of claims 8 to 9 in forensic rapid individual identification.

Images