Multiplex amplification kit based on DIP-TriSNP linked genetic markers and its application

Abstract

The present invention relates to the field of forensic medicine, and specifically provides a composite amplification kit based on DIP-TriSNP linked genetic markers and applications. The composite amplification kit provided by the present invention includes a composite amplification detection system, including a composite PCR amplification primer mixture and a single base extension reaction primer mixture for amplifying 14 autosomal DIP-TriSNP sites, 1 Amel gene locus and 1 Y chromosome DIP-SNP site; the composite PCR amplification primers for each site include two specific upstream primers and a universal downstream primer. The DIP-TriSNP composite amplification detection kit provided by the present invention has high sensitivity, strong species specificity, and strong personal recognition ability; it can reduce the number of one-time detection sites, make the detection method simpler, and shorten the detection time.

Description

Technical Field

[0001] The invention belongs to the field of forensic medicine, and in particular relates to a composite amplification kit based on DIP-TriSNP linked genetic markers and an application thereof. Background Art

[0002] Forensic genetic markers are often used in forensic cases. They can distinguish the typing of each individual from the mixed spots of two or even more individuals. Currently, the commonly used forensic genetic markers mainly include short tandem repeats (STR), insertion / deletion polymorphisms (DIP), and single nucleotide polymorphisms (SNP). Among them, STR has developed into the mainstream genetic marker used in forensic DNA typing due to its high sensitivity, high identification ability, high species specificity, high accuracy of results, and easy standardization. However, due to the "tandem repeat" attribute of STR itself, replication slippage often occurs during STR amplification, resulting in the appearance of shadow peaks. The existence of shadow peaks may make it difficult to distinguish between minor components and shadow peaks, and STR has the problem of unbalanced allele amplification. Compared with STR, DIP and SNP have the advantages of small amplification fragments, low mutation rate, wide distribution, no artificial pseudo-peaks such as shadow peaks, and can predict ethnic origin or phenotype. However, most DIPs and SNPs are di-allelic in the population, so their polymorphism is not as good as STR, which requires many genetic markers to achieve the identification capabilities required in forensic medicine. In the SNPs of the human genome, in addition to conventional di-allelic SNPs, there are also many tri-allelic SNPs. Phillips et al. used SNaPshot technology to construct a composite system containing 27 tri-allelic SNPs and 2 quad-allelic SNPs. Its polymorphism is greatly improved compared to di-allelic SNPs, and it has good results in the accuracy of analyzing mixed spots.

[0003] In order to solve the problems existing in the analysis of mixed samples with existing genetic markers and improve the polymorphism of genetic markers, domestic and foreign scholars often choose to link multiple genetic markers, such as microhaplotype, DIP-microhaplotype, and DIP-SNP. At present, the typing method of microhaplotype genetic markers mainly relies on high-throughput sequencing technology. However, due to factors such as high prices, capillary electrophoresis (CE), which is more popular and less expensive, is often used to detect linked genetic markers in the study of mixed plaque analysis. Among them, the DIP-SNP linked genetic marker uses SNaPshot technology on CE to study mixed plaque analysis. The linked SNPs are all dialleles, and the polymorphism is lower than that of trialleles, so that more sites are needed in the analysis of mixed plaque results to obtain better results. However, when the CE platform is detected, the number of sites detected at one time is limited, and the DIP-SNP linked genetic marker has only 4 alleles. Summary of the invention

[0004] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise. Numerical ranges recited by endpoints include all numbers and fractions within the corresponding ranges, as well as the recited endpoints.

[0005] In view of the problems existing in the prior art, the present invention provides a composite amplification kit based on DIP-TriSNP linked genetic markers and its application, which can reduce the number of one-time detection sites, make the detection method simpler and shorten the detection time; in forensic mixed spot analysis, the sensitivity and specificity are high and the detection results are accurate.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] On the one hand, the present invention provides a composite amplification detection system based on DIP-TriSNP linked genetic markers, comprising a composite PCR amplification primer mixture and a single base extension reaction primer mixture for amplifying 14 autosomal DIP-TriSNP sites, 1 Amel gene locus and 1 Y chromosome DIP-SNP site; the composite PCR amplification primers for each site include two specific upstream primers and a universal downstream primer.

[0008] Specifically, the Amel gene locus is a sex identification locus.

[0009] Preferably, the rs numbers of the 14 autosomal DIP-TriSNP sites and 1 Y chromosome DIP-SNP site are rs71909366-rs9975846, rs72010079-rs11632224, rs3975820-rs2864789, rs142533588-rs13316343, rs148039798-rs7740297, rs35808370-rs1917626, rs780425 73-rs12482290, rs149419997-rs4942957, rs139633194-rs1823835, rs10598574-rs640711, rs148751034-rs1908166, rs146904464-rs3910500, rs75337291-rs3915403, rs72381597-rs12103203 and rs2032678-rs866861925.

[0010] Specifically, the DIP sites and SNP site compositions of the 15 DIP-TriSNP sites and the chromosome numbers on which they are located are shown in Table 1 (the SNP sites are named after the rs numbers in the SNP database of the National Center for Biotechnology Information of the United States);

[0011] Table 1 Composition of DIP sites and SNP sites of 15 DIP-TriSNP sites

[0012] Location rs number mh21FQ-03 rs71909366 / rs9975846 mh0YFQ-02 rs2032678 / rs866861925 mh15FQ-05 rs72010079 / rs11632224 mh03FQ-08 rs3975820 / rs2864789 mh03FQ-06 rs142533588 / rs13316343 mh06FQ-23 rs148039798 / rs7740297 mh08FQ-03 rs35808370 / rs1917626 mh21FQ-02 rs78042573 / rs12482290 mh13FQ-02 rs149419997 / rs4942957 mh06FQ-21 rs139633194 / rs1823835 mh01FQ-04 rs10598574 / rs640711 mh11FQ-02 rs148751034 / rs1908166 mh18FQ-01 rs146904464 / rs3910500 mh19FQ-01 rs75337291 / rs3915403 mh16FQ-03 rs72381597 / rs12103203

[0013] Preferably, the composite PCR amplification primer mixture comprises 48 amplification primers, and the nucleotide sequence of each primer is shown as SEQ ID No.1 to SEQ ID No.48 respectively.

[0014] Specifically, the sequences of the 16 sets of primers used to amplify the 15 DIP-TriSNP sites and the Amel locus and the concentration of each set of primers in the composite amplification system are shown in Tables 2 and 3;

[0015] Table 2 Universal primers, L-type primers and their concentrations for 15 DIP-TriSNP loci and Amel locus

[0016]

[0017]

[0018] Table 3 Universal primers, S-type primers and their concentrations for 15 DIP-TriSNP loci and Amel locus

[0019]

[0020]

[0021] Specifically, the primer concentrations described in Tables 2 and 3 refer to the sum of the concentrations of the universal primer, L-type primer and S-type primer in each set of primers, that is, at the specific concentrations described in Tables 2 and 3 above, the amplification efficiency of each site is kept as consistent as possible.

[0022] Preferably, the single base extension reaction primer mixture comprises 16 primers, and the nucleotide sequence of each primer is shown in SEQ ID No.49 to SEQ ID No.64 respectively.

[0023] Specifically, the sequences of the single base extension reaction primers and the concentration of each set of primers in the composite system are shown in Table 4;

[0024] Table 4 Single base extension primers and concentrations for 15 DIP-TriSNP sites and Amel locus

[0025]

[0026]

[0027] Specifically, at the specific concentrations described in Table 4 above, the amplification efficiency of each site is kept as consistent as possible.

[0028] On the other hand, the present invention provides a use of the above-mentioned composite amplification detection system in the preparation of a composite amplification detection kit.

[0029] In another aspect, the present invention provides a composite amplification detection kit based on DIP-TriSNP linked genetic markers, comprising the above composite amplification detection system.

[0030] Preferably, the composite amplification detection kit further comprises a composite amplification reaction reagent.

[0031] Further preferably, the composite amplification reaction reagents include but are not limited to PCR buffer solution, DNA template, and ddH2O.

[0032] Specifically, the amount of the composite amplification reaction reagent is shown in Table 5;

[0033] Table 5 Amplification system

[0034]

[0035] Wherein, the Buffer is a buffer solution of the amplification system, and the Pimer mix is ​​a mixture of primers shown in Table 2 and Table 3.

[0036] Preferably, the composite amplification program is: 95°C, 15 min; 94°C, 30 s, 60°C, 90 s, 72°C, 1 min, 32 cycles; 68°C, 15 min; 4°C, storage.

[0037] Preferably, the composite amplification detection kit further comprises an amplification product purification reagent.

[0038] Further preferably, the amplification product purification reagent includes but is not limited to shrimp alkaline phosphatase, shrimp alkaline phosphatase buffer, exonuclease, and exonuclease buffer.

[0039] Specifically, the amount of the amplification product purification reagent added is shown in Table 6;

[0040] Table 6 Sample volume of amplification product purification reagent

[0041]

[0042] Wherein, the rSAP is shrimp alkaline phosphatase, rSAP Buffer is shrimp alkaline phosphatase buffer, ExoⅠ is nuclease exonuclease, and ExoⅠBuffer is nuclease exonuclease buffer.

[0043] Preferably, the purification procedure is: 37°C, 1 h; 95°C, 15 min; 4°C, storage.

[0044] Preferably, the composite amplification detection kit further comprises a single base extension reaction reagent for amplifying the above 15 DIP-TriSNP sites and Amel.

[0045] Preferably, the single base extension reaction reagent comprises a buffer.

[0046] Preferably, the composite amplification detection kit also includes reagents commonly used in the SNaPshot system.

[0047] Further preferably, the commonly used reagents of the SNaPshot system include but are not limited to ddH2O and SNaPshot reaction buffer.

[0048] Specifically, the amount of sample added in the SNaPshot system is shown in Table 7;

[0049] Table 7 SNaPshot reaction system

[0050]

[0051] Wherein, the SNaPshot Reaction Mix is ​​a SNaPshot reaction buffer, and the Pimer mix is ​​a mixture of primers shown in Table 4.

[0052] Preferably, the SNaPshot reaction procedure is: 96°C, 10s, 50°C, 5s, 60°C, 30s, 32 cycles; 4°C, storage.

[0053] Preferably, the composite amplification detection kit further comprises a purification reagent for the SNaPshot reaction product.

[0054] Further preferably, the purification reagents also include but are not limited to H2O and shrimp alkaline phosphatase.

[0055] Specifically, the amount of each reagent added in the SNaPshot product purification system is shown in Table 8 below:

[0056] Table 8 SNaPshot product purification system

[0057]

[0058] Wherein, the rSAP is shrimp alkaline phosphatase.

[0059] Preferably, the purification procedure is: 37°C, 1 h; 95°C, 15 min; 4°C, storage.

[0060] In another aspect, the present invention further provides a method for mixed plaque analysis using the above-mentioned composite amplification detection kit, comprising the following steps:

[0061] S1, extracting the DNA of the sample to be tested as an amplification template;

[0062] S2, using a composite PCR amplification primer mixture and a composite amplification reaction reagent to perform in-tube composite PCR amplification on the DNA extracted in step A to obtain a composite amplification product;

[0063] The program of the composite PCR amplification is: 95°C, 15 minutes; 94°C, 30 seconds, 60°C, 90 seconds, 72°C, 1 minute, 32 cycles; 68°C, 15 minutes;

[0064] S3, purifying the composite amplification product in step S2, using the purified product as a template and performing multiple single base extension reactions in a single tube using a single base extension reaction primer mixture and a single base extension reaction reagent to obtain multiple single base extension products;

[0065] The purification step comprises: taking 3.8 μL of the composite amplification product, adding 1.2 μL of shrimp alkaline phosphatase, 1 μL of shrimp alkaline phosphatase buffer, 0.3 μL of exonuclease, 1 μL of exonuclease buffer and 2.7 μL of deionized water, and mixing them to perform a purification reaction, wherein the reaction procedure is 37° C. for 60 minutes, 95° C. for 15 minutes, and storage at 4° C.;

[0066] The steps of the multiple single base extension reaction include: taking 2 μL of the purified product, adding 2 μL of buffer, a single base extension reaction primer mixture and 0.5 μL of deionized water, and mixing them to perform a single base extension reaction, wherein the reaction procedure is 96° C., 10 seconds, 50° C., 5 seconds, 60° C., 30 seconds, and 32 cycles; and storing at 4° C.;

[0067] S4, purifying the multiple single base extension products of step S3, then performing capillary electrophoresis, and analyzing the mixed spots according to the electrophoresis results;

[0068] The purification step includes: taking 5 μL of multiple single base extension products, adding 1 μL of shrimp alkaline phosphatase and mixing to perform purification reaction, and the reaction procedure is 37°C, 60 minutes, 95°C, 15 minutes, and 4°C storage.

[0069] Preferably, the conditions of capillary electrophoresis in step S4 include: pre-electrophoresis voltage 15 KV, time 60 s; injection voltage 2 KV, time 15 s; electrophoresis voltage 15 KV, electrophoresis time 1200 s.

[0070] Specifically, 8.85 μL Hi-Di formamide, 1 μL purified product and 0.15 μL GeneScanTM 120LIZSize Standards internal standard were mixed; data were collected using Data Collection v3.0 software; and electrophoresis data were analyzed using GeneMapper ID-Xv1.4 software.

[0071] Preferably, the identification criteria for analyzing the mixed spots in step S4 are as follows: The identification criteria for analyzing the mixed spots in step S4 include: (1) the number of allele peaks at each site is more than 2; (2) for a single tube reaction, the peak height ratio of the alleles at each site is less than or equal to 60%.

[0072] Specifically, the identification criteria for mixed spots are as follows: (1) Determine whether it is a mixed spot: check the number of allele peaks at each site. If there are more than two peaks, especially when there are more than two peaks at multiple sites at the same time, it can be determined as a mixed spot; check the relative peak height of the spectrum of each site. Under normal circumstances, the peak height ratio of the two peaks should be greater than 60%. If it does not meet this rule, it indicates the existence of mixed spots; (2) Statistical analysis: In addition to considering the number and type of alleles, the peak height and peak area of ​​each allele are also considered to exclude some impossible genotype combinations and screen out reasonable phenotypic combinations. Then refer to the genotype of the sample for comparison and conduct a comprehensive likelihood analysis.

[0073] In yet another aspect, the present invention further provides an application of the above-mentioned composite amplification detection kit in the field of forensic identification and forensic detection.

[0074] Specifically, the applications in the field of forensic identification and detection include but are not limited to individual identification, case investigation and mixed spot identification in the field of forensic identification and detection.

[0075] Compared with the prior art, the present invention has the following beneficial effects:

[0076] The DIP-TriSNP site and the detection system containing the DIP-TriSNP site in the present invention are suitable for forensic mixed spot analysis, and the number of alleles can reach 6, which can reduce the number of one-time detection sites, make the detection method simpler, and shorten the detection time; and the DIP-TriSNP site and the detection system containing the DIP-TriSNP site in the present invention have high sensitivity and specificity in forensic mixed spot analysis, and the detection result is accurate. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 This is the SNaPshot result diagram of the male sample at 16 sites in Example 1.

[0078] Figure 2 This is the SNaPshot result diagram of the female samples at 16 sites in Example 1.

[0079] Figure 3 This is the Sanger sequencing result of the S-type amplification product of the mh21FQ-02 site. DETAILED DESCRIPTION

[0080] The present invention is described below through specific embodiments to make the technical solution of the present invention easier to understand and grasp, but the present invention is not limited thereto, and the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments.

[0081] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0082] Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of this application. The experimental methods described in the following embodiments are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0083] Example 1 Construction of microhaplotype complex amplification system

[0084] 1. Site selection

[0085] The criteria for site selection are as follows:

[0086] (1) The allele insertion or deletion at the DIP site is 3-10 bp;

[0087] (2) the average heterozygosity of DIP and SNP loci was ≥ 0.3;

[0088] (3) the distance between the DIP and SNP sites is no more than 200 bp;

[0089] (4) The composite PCR fragment does not exceed 230 bp;

[0090] (5) The SNP locus is tri-allelic in East Asian populations;

[0091] (6) If multiple loci are located on the same chromosome, the distance between the loci should be greater than 10 Mbp.

[0092] Fifteen loci were screened, and the specific locus information is shown in Table 9, which includes the information of 15 DIP-TriSNP loci and Amel locus, including the locus name, chromosome number, rs number of DIP or SNP in the human whole genome Dec.2013 (GRCh38 / hg38 version) reference genome, starting position and reference typing.

[0093] Among them, "mh" in the site name represents microhaplotype, and the two digits after mh represent the chromosome where the microhaplotype locus is located. Taking the mh21FQ-03 site as an example, "21" represents that it is located on chromosome 21, "FQ" represents the naming code, and "03" represents that the site is the third site found on chromosome 3 in the present invention.

[0094] Table 9 Information of 15 DIP-TriSNP sites and Amel locus

[0095]

[0096] 2. Primer Design

[0097] The primer design requirements for the above selected sites for composite PCR are as follows: the primer length is between 18-35bp; the Tm values ​​of the primers of each locus are as consistent as possible, and the Tm value mainly depends on the software design results of Primer; the primers cannot form dimers or hairpin structures; the PCR length is controlled within 230bp; the amplification product obtained by the primers in PCR is single.

[0098] Table 2 and Table 3 are the universal primers, L-type allele primers and S-type allele primers of 15 DIP-TriSNP sites and Amel locus and their respective concentration information and amplification product fragment length information. Under the specific concentrations of Table 2 and Table 3, the amplification efficiency of each site is kept consistent as much as possible.

[0099] 3. Single locus PCR amplification detection

[0100] The designed primers were subjected to single locus PCR amplification and agarose gel electrophoresis detection, and single locus amplification primers with good amplification efficiency and single target band were selected for the next step of composite amplification.

[0101] 4. Multiplex PCR Amplification

[0102] The primers screened after the above single sample PCR amplification are mixed into a reaction system. The most appropriate primer concentration is selected under the condition of ensuring a certain template concentration, so that each locus in the composite system can be optimally amplified as much as possible.

[0103] The amount of each reagent added in the composite system is shown in Table 5; wherein the Buffer is the buffer of the amplification system, and the Pimer mix is ​​a mixture of primers shown in Tables 2 and 3.

[0104] The multiplex PCR program was performed according to the conditions shown in Table 10:

[0105] Table 10 Amplification program

[0106]

[0107] 5. Purification of Composite PCR Products

[0108] The amount of each reagent added for the composite PCR product purification reaction is shown in Table 6; wherein, the rSAP is shrimp alkaline phosphatase, rSAP Buffer is shrimp alkaline phosphatase buffer, ExoⅠ is nuclease exonuclease, and ExoⅠBuffer is nuclease exonuclease buffer.

[0109] The purification reaction was carried out under the following conditions:

[0110] 37°C, 1h;

[0111] 95℃, 15min;

[0112] 4℃,∞.

[0113] 6.SNaPshot reaction

[0114] The single base extension primers of the DIP-TriSNP loci selected above were mixed into a reaction system. The most suitable primer concentration was selected under the condition of ensuring a certain DNA concentration, so that each locus in the composite system could achieve optimal amplification as much as possible. Table 4 shows the concentration information and amplification product fragment length of the single base extension primers of 15 DIP-TriSNP loci and Amel locus in the amplification system.

[0115] The amount of each reagent added in the SNaPshot reaction system is shown in Table 7; wherein the SNaPshot ReactionMix is ​​the SNaPshot reaction buffer, and the Pimer mix is ​​the mixture of primers shown in Table 4.

[0116] The SNaPshot reaction procedure was performed under the following conditions:

[0117]

[0118] 7. Purification of SNaPshot Products

[0119] The amount of each reagent added in the SNaPshot product purification reaction is shown in Table 8; wherein the rSAP is shrimp alkaline phosphatase.

[0120] The purification reaction was carried out under the following conditions:

[0121] 37°C, 1h;

[0122] 95℃, 15min;

[0123] 4℃,∞.

[0124] The SNaPshot results of 16 loci including the Amel locus are as follows Figure 1 and Figure 2 As shown, all loci were amplified.

[0125] Example 2 Determination of Population Frequency and Forensic Parameters

[0126] Since the DIP-TriSNP sites provided by the present invention are all newly designed, there is no corresponding frequency information that can be used to analyze mixed spots when applied to the kit, so the frequencies of these micro-haplotypes need to be calculated first.

[0127] (1) DNA extraction: The whole genomic DNA of the sample was extracted using the Biospin whole blood genomic DNA extraction kit.

[0128] (2) Performing composite PCR amplification on the extracted DNA according to the composite amplification system provided in Example 1.

[0129] (3) The obtained PCR products were subjected to capillary electrophoresis, and the data were collected using Data Collection v3.0 software; the electrophoresis data were analyzed using GeneMapper ID-X v1.4 software.

[0130] (4) The locus is determined based on the position of the peak in the spectrum, the DIP typing is determined based on the presence or absence of allele peaks in the L-type reaction and the S-type reaction, and the SNP typing of the locus is determined based on the color of the peak. The DIP typing and SNP typing are combined to obtain the haplotype typing of the individual at the locus.

[0131] (5) Use STRAF online software to calculate forensic parameters such as haplotype frequencies.

[0132] In this embodiment, 50 unrelated samples were selected from the Han population in Sichuan, and the haplotypes appearing on each chromosome and the corresponding frequencies are shown in Table 11 below.

[0133] Table 11 Haplotypes and corresponding frequencies of 14 autosomal DIP-TriSNP loci in Sichuan Han samples

[0134]

[0135]

[0136] The results of forensic parameters calculated by STRAF online software are shown in Table 12 below.

[0137] Table 12 Forensic parameters of 14 autosomal DIP-TriSNP loci in the Han population of Sichuan

[0138]

[0139] According to the cumulative personal identification (CPD) calculation formula:

[0140] CPD=1-(1-PD1)(1-PD2)(1-PD3)……(1-PD4)

[0141] It can be obtained that CPD≈0.999999999;

[0142] According to the cumulative probability of exclusion (CPE) calculation formula:

[0143] CPE=1-(1-PE1)(1-PE2)(1-PE3)……(1-PE14)

[0144] It can be obtained that CPE≈0.97851555.

[0145] According to the above results, the composite amplification system provided in Example 1 has good polymorphism in the Han nationality in Sichuan. Compared with conventional STR kits, while ensuring personal identification ability, the number of loci is small and the amplified fragment is smaller, which can play an auxiliary role in forensic personal identification and is of great significance in the analysis of forensic mixed spots.

[0146] Test Example 1 Sensitivity Experiment

[0147] The composite amplification system provided in Example 1 was subjected to sensitivity detection experiments at three standard template concentrations of 10 ng, 1 ng and 0.1 ng. It was found that complete typing of the sample could be obtained at DNA template amounts of 10 ng and 1 ng. When the template amount was 0.1 ng, allele loss occurred. It is recommended that the composite system can detect DNA samples with a content of more than 1 ng.

[0148] Test Example 2 Species-specific Experiment

[0149] The composite amplification system provided in Example 1 was used to perform composite PCR amplification on DNA of six animals, namely, mouse, rabbit, chicken, duck, cattle and fish. Fluorescent signals of different loci were found in the typing maps of the six animals, indicating that the composite system was species-specific.

[0150] Test Example 3: Accuracy and consistency experiment

[0151] The product after composite amplification in Example 1 was subjected to Sanger sequencing. Figure 3 This is the Sanger sequencing result of the S-type amplification product of the mh21FQ-02 locus. Compared with the sequence in the human genome Dec. 2013 (GRCh38 / hg38 version) reference genome, the results are consistent, indicating that the composite system has high accuracy.

[0152] The samples were randomly selected and tested three times using the composite amplification system provided in Example 1, and the typing results were consistent, indicating that the composite system has consistency.

[0153] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A composite amplification kit based on DIP-TriSNP linked genetic markers, characterized in that: The composite amplification kit includes a composite PCR amplification primer mixture and a single base extension reaction primer mixture for amplifying 14 autosomal DIP-TriSNP sites, 1 Amel gene locus and 1 Y chromosome DIP-SNP site; the composite PCR amplification primers for each site include two specific upstream primers and one universal downstream primer; The rs numbers of the 14 autosomal DIP-TriSNP sites and 1 Y chromosome DIP-SNP site are rs71909366-rs9975846, rs72010079-rs11632224, rs3975820-rs2864789, rs142533588-rs13316343, rs148039798-rs7740297, rs35808370-rs1917626, rs78042573-rs12482290, rs14941999 7-rs4942957, rs139633194-rs1823835, rs10598574-rs640711, rs148751034-rs1908166, rs146904464-rs3910500, rs75337291-rs3915403, rs72381597-rs12103203 and rs2032678-rs866861925; the composite PCR amplification primer mixture comprises 48 amplification primers, and the nucleotide sequences of each amplification primer are shown in SEQ ID No.1 to SEQ ID No.48, respectively; the single base extension reaction primer mixture comprises 16 primers, and the nucleotide sequences of each primer are shown in SEQ ID No.49 to SEQ ID No.64, respectively.

2. The composite amplification detection kit according to claim 1, characterized in that: The composite amplification detection kit also includes a composite amplification reaction reagent, an amplification product purification reagent and a single base extension reaction reagent.

3. The composite amplification detection kit according to claim 2, characterized in that: The composite amplification reaction reagents include PCR buffer solution and DNA template; the amplification product purification reagents include shrimp alkaline phosphatase, shrimp alkaline phosphatase buffer, nuclease exonuclease, and nuclease exonuclease buffer; the single base extension reaction reagents include buffer.

4. A method for mixed plaque analysis using the composite amplification detection kit as claimed in claim 3, characterized in that: The method comprises the following steps: S1, extracting the DNA of the sample to be tested as an amplification template; S2, using the composite PCR amplification primer mixture and composite amplification reaction reagent in the composite amplification detection kit as claimed in claim 3 to perform composite PCR amplification on the sample DNA to be detected extracted in S1 to obtain a composite amplification product; The program of the composite PCR amplification is: 95°C, 15 minutes; 94°C, 30 seconds, 60°C, 90 seconds, 72°C, 1 minute, 32 cycles; 68°C, 15 minutes; S3, purifying the composite amplification product in step S2, using the purified product as a template and performing a single-tube multiple single-base extension reaction using a single-base extension reaction primer mixture and a single-base extension reaction reagent in the composite amplification detection kit as claimed in claim 3 to obtain multiple single-base extension products; The purification step comprises: taking 3.8 μL of the composite amplification product, adding 1.2 μL of shrimp alkaline phosphatase, 1 μL of shrimp alkaline phosphatase buffer, 0.3 μL of exonuclease, 1 μL of exonuclease buffer and 2.7 μL of deionized water, and mixing them to perform a purification reaction, wherein the reaction procedure is 37° C. for 60 minutes, 95° C. for 15 minutes, and storage at 4° C.; The steps of the multiple single base extension reaction include: taking 2 μL of the purified product, adding 2 μL of buffer, a single base extension reaction primer mixture and 0.5 μL of deionized water, and mixing them to perform a single base extension reaction, wherein the reaction procedure is 96° C., 10 seconds, 50° C., 5 seconds, 60° C., 30 seconds, and 32 cycles; and storing at 4° C.; S4, purifying the multiple single base extension products of step S3, then performing capillary electrophoresis, and analyzing the mixed spots according to the electrophoresis results; The purification step comprises: taking 5 μL of multiple single-base extension products, adding 1 μL of shrimp alkaline phosphatase and mixing to perform purification reaction, the reaction procedure is 37°C, 60 minutes, 95°C, 15 minutes, and storage at 4°C; The conditions of the capillary electrophoresis include: pre-electrophoresis voltage 15KV, time 60s; injection voltage 2KV, time 15s; electrophoresis voltage 15KV, electrophoresis time 1200s.

5. The method according to claim 4, characterized in that: The identification criteria for analyzing mixed spots in step S4 include: (1) the number of allele peaks at each site is more than 2; (2) for a single tube reaction, the peak height ratio of the two allele peaks at each site is less than or equal to 60%.

6. Use of the composite amplification detection kit according to any one of claims 1 to 3 in the field of forensic identification and forensic detection.

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