A fluorescent multiplex amplification detection kit for human autosomal InDel sites and application thereof

Abstract

The application belongs to the technical field of gene detection, and relates to a gene detection kit and application thereof. Specifically, the application relates to a kit for simultaneously detecting 31 human autosomal InDel sites and 1 sex identification gene and application thereof. The kit contains 31 pairs of specific amplification primers of autosomal InDel sites and 1 specific amplification primer of a sex identification gene, is suitable for InDel typing of conventional biological samples and degraded biological samples, and can be applied to human individual identification and parentage identification.

Description

Technical Field

[0001] This invention belongs to the field of gene detection technology, specifically relating to a kit for simultaneously detecting 31 human autosomal InDel loci and 1 sex identification gene and its application. Background Technology

[0002] In the field of forensic science, DNA typing techniques are widely used in judicial practices such as individual identification and paternity testing. Among these, short tandem repeat (STR) typing is currently the most widely applied technique. STRs are microsatellite DNA sequences, typically consisting of 2-6 base pairs as the core repeat unit. With the widespread use of STR loci in forensic DNA typing, the limitations of these genetic markers have become increasingly apparent and have received ongoing attention. For example, due to the relatively long length of STR amplicon, their detection efficiency in degraded samples is low, making it difficult to type degraded biological samples; STRs have a high mutation rate, making them prone to mutations during downward inheritance, leading to phenomena that do not conform to the laws of inheritance in paternity testing. Currently, the academic community is exploring different methods to optimize STR detection or developing new genetic marker types to replace the STR typing system and apply them to forensic DNA typing through research on different genetic polymorphic markers.

[0003] Insertion / deletion polymorphism (InDel) refers to genetic variations at a specific locus in the genome caused by the insertion or deletion of DNA fragments of varying lengths, exhibiting characteristics of both STRs and SNPs. Based on the sequence characteristics of InDel insertions, InDels in the genome can be divided into three main categories: the first category consists of single-base or random DNA sequence insertions / deletions; the second category consists of amplifications of single or 2-15 base pairs as units; and the third category consists of transposon insertions. The first category of InDels is presumed to be caused by random DNA sequence insertions or deletions, and therefore may have a lower mutation rate. The second category of InDels is generally considered to be caused by replication slippage leading to the replication or deletion of repetitive units, similar to the mutation mechanism of STRs, and therefore may have a higher mutation rate; to some extent, STRs also belong to the InDel genetic marker category. The third category of InDels consists of random insertions of transposon elements; due to their relatively long length, their application in forensic medicine is relatively limited. Therefore, InDel genetic markers have greater significance in individual identification and paternity testing.

[0004] Currently, commercially available InDel kits are available for DNA detection in biological assays. However, the selected InDel loci are dialleles, meaning there are only two alleles, resulting in low polymorphism and making these kits unsuitable for forensic practice. Some researchers have attempted to improve the discriminative power of InDel kits by increasing the number of detection sites; however, due to the limited number of sites that a multiplex amplification system can simultaneously detect, simply increasing the number of sites is insufficient to significantly improve the efficiency of InDel kits.

[0005] Based on the 'secondary mutation' hypothesis of random InDel sequences proposed in previous research of this invention, that is, a second insertion or deletion can occur at an insertion / deletion site, thus generating new alleles; through analysis of deep sequencing data from the 1000-person genome, more than 3000 multi-allelic InDel sequences with random insertion / deletion sequences were screened, further confirming the aforementioned hypothesis. This application carefully screened and identified InDel sites in the human genome that contain multiple alleles and have high polymorphism in the Chinese population, and established a detection and analysis kit that is accurate, convenient, and has high identification efficiency. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and limitations of existing technologies and provide a kit with high identification efficiency, suitable for the Chinese population, and applicable to the identification and analysis of routine biological samples and degraded biological samples. Specifically, it provides a human autosomal InDel site fluorescence multiplex amplification kit and its application.

[0007] This invention's kit contains 31 relatively balanced InDel loci and 1 sex identification gene locus, employing a five-color fluorescently labeled multiplex amplification system. The 31 InDel loci were screened based on population genetic data from East Asia and further validated in the Chinese population; each locus contains 3 or more alleles, exhibiting high identification efficiency. The amplification products from this kit are short fragments, all less than 225 bp, suitable for DNA typing of both routine and degraded biological samples.

[0008] To achieve the objectives of this invention, the following technical solution is adopted:

[0009] The first aspect of this invention provides a gene detection kit, which is a human autosomal InDel site fluorescence multiplex amplification kit.

[0010] The gene detection kit provided by this invention contains 31 pairs of specific amplification primers for autosomal InDel loci, and 1 pair of specific amplification primers for sex identification genes.

[0011] As a preferred technical solution, the autosomal InDel loci are the following loci: HID1-1 (rs60893206), HID1-2 (rs58568060), HID1-3 (rs1553161226), HID1-4 (rs34314833), HID2-1 (rs77948797), HID3-1 (rs139809929), HID3-2 (rs386670017), HID4-1 ( rs3077701), HID4-2 (rs70949989), HID5-1 (rs200753977), HID6-1 (rs10635385), HID6-2 (rs15542199 17), HID7-1(rs58558819), HID7-2(rs6150152), HID7-3(rs112257258), HID8-1(rs10596541), HID8-2( rs59118283), HID9-1(rs386737038), HID9-2(rs138815275), HID11-1(rs11271121), HID11-2(rs6657 2312), HID11-3(rs1555121221), HID12-1(rs6144790), HID13-1(rs386768785), HID14-1(rs33958617) HID14-2 (rs58233606), HID16-1 (rs1383237378), HID18-1 (rs772191138), HID19-1 (rs113712314), HID20-1 (rs11467514), and HID22-1 (rs758153887), where the rs number in parentheses is the locus number in the dbSNP database; the sex determination gene is the Amelogenin gene.

[0012] As a further preferred technical solution, the PCR amplification products of the specific amplification primers are all less than 225 bp in length.

[0013] As a further preferred technical solution, the specific amplification primer sequences are as follows: HID1-1, SEQ ID NO.1-2; HID1-2, SEQ ID NO.3-4; HID1-3, SEQ ID NO.5-6; HID1-4, SEQ ID NO.7-8; HID2-1, SEQ ID NO.9-10; HID3-1, SEQ ID NO.11-12; HID3-2, SEQ ID NO.13-14; HID4-1, SEQ ID NO.15-16; HID4-2, SEQ ID NO.17-18; HID5-1, SEQ ID NO.19-20; HID6-1, SEQ ID NO.21-22; HID6-2, SEQ ID NO.23-24; HID7-1, SEQ ID NO.25-26; HID7-2, SEQ ID NO.27-28; HID7-3 ...29-20; HID1-1, SEQ ID NO.21-22; HID6-2, SEQ ID NO.29-20; HID7-1, SEQ ID NO.21-22; HID6-2, SEQ ID NO.29-20; HID7-1, SEQ ID NO.29-30; HID8-1, SEQ ID NO.31-32; HID8-2, SEQ ID NO.33-34; HID9-1, SEQ ID NO.35-36; HID9-2, SEQ ID NO.37-38; HID11-1, SEQ ID NO.39-40; HID11-2, SEQ ID NO.41-42; HID11-3, SEQ ID NO.43-44; HID12-1, SEQ ID NO.45-46; HID13-1, SEQ ID NO.47-48; HID14-1, SEQ ID NO.49-50; HID14-2, SEQ ID NO.51-52; HID16-1, SEQ ID NO.53-54; HID18-1, SEQ ID NO.55-56; HID19-1, SEQ ID NO.57-58; HID20-1, SEQ ID NO.59-60; HID22-1, SEQ ID NO.61-62; Amelogenin, SEQ ID NO.63-64.

[0014] As a further preferred technical solution, the reaction concentrations of the specific amplification primers are as follows: HID1-1, 0.48 μM; HID1-2, 0.48 μM; HID1-3, 0.48 μM; HID1-4, 0.48 μM; HID2-1, 0.48 μM; HID3-1, 0.48 μM; HID3-2, 0.08 μM; HID4-1, 0.48 μM; HID4-2, 0.48 μM; HID5-1, 0.48 μM; HID6-1, 0.48 μM; HID6-2, 0.12 μM; HID7-1, 0.24 μM; HID7-2, 0.12 μM; HID7-3, 0.24 μM; HID8-1 , 0.16μM; HID8-2, 0.24μM; HID9-1, 0.12μM; HID9-2, 0.48μM; HID11-1, 0.12 μM; HID11-2, 0.48μM; HID11-3, 0.48μM; HID12-1, 0.16μM; HID13-1, 0.48μM ; HID14-1, 0.16μM; HID14-2, 0.16μM; HID16-1, 0.16μM; HID18-1, 0.08μM; HID19-1, 0.12μM; HID20-1, 0.12μM; HID22-1, 0.96μM; Amelogenin, 0.48μM.

[0015] The primer sequences for the above 32 loci and the preferred concentrations of the primers in the amplification system are shown in Table 1.

[0016] Table 1 Primers and their concentrations for each gene locus

[0017]

[0018]

[0019] As a preferred technical solution, the specific amplification primers are divided into four groups, as follows: the first group consists of HID3-2, HID7-1, HID8-1, HID12-1, HID14-1, HID14-2, HID16-1, and HID18-1; the second group consists of HID6-2, HID7-2, HID7-3, HID8-2, HID9-1, HID11-1, HID19-1, and HID20-1. The third group consists of HID1-1, HID1-4, HID2-1, HID3-1, HID9-2, HID11-3, and HID22-1; the fourth group consists of Amelogenin, HID1-2, HID1-3, HID4-1, HID4-2, HID5-1, HID6-1, HID11-2, and HID13-1; in each primer pair, the 5' end of one primer is labeled with a fluorescent dye.

[0020] As a further preferred technical solution, the four sets of primers are labeled with blue, green, yellow and red fluorescent dyes, respectively, with each set using a different fluorescent label.

[0021] As a further preferred technical solution, the primers are labeled using the following method: the blue label can be 5-FAM (5-carboxyfluorescein), 6-FAM (6-carboxyfluorescein), or a fluorescein molecule with a similar spectrum; the green label can be HEX (hexachloro-6-methylfluorescein), JOE (6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein succinimide), or a fluorescein molecule with a similar spectrum; the yellow label can be TAMRA (carboxytetramethylrhodamine), or a fluorescein molecule with a similar spectrum; and the red label can be ROX (carboxy-X-rhodamine), or a fluorescein molecule with a similar spectrum.

[0022] As a further preferred technical solution, the specific amplification primers are grouped and labeled as follows: Fluorescent dye molecules 6-FAM, HEX, TAMRA, and ROX are used to label the primers; wherein: 6-FAM labels HID3-2, HID7-1, HID8-1, HID12-1, HID14-1, HID14-2, HID16-1, and HID18-1; HEX labels HID6-2, HID7-2, HID7-3, and HID18-1. D8-2, HID9-1, HID11-1, HID19-1, HID20-1; TAMRA-labeled HID1-1, HID1-4, HID2-1, HID3-1, HID9-2, HID11-3, HID22-1; ROX-labeled Amelogenin, HID1-2, HID1-3, HID4-1, HID4-2, HID5-1, HID6-1, HID11-2, HID13-1.

[0023] As a further preferred technical solution, the kit contains 2×Multiplex PCR Master Mix; 5×Primer Mixture; BSA; Hot-start Taq Enzyme; ddH2O.

[0024] The amplification system of the kit can achieve good results on various reaction thermal cyclers using the following procedure: incubate at 95°C for 5 minutes; incubate at 94°C for 30 seconds, incubate at 60°C for 90 seconds, incubate at 70°C for 30 seconds, repeat this step 29 times; incubate at 60°C for 35 minutes; incubate at 4°C.

[0025] A second aspect of this invention provides an application of a gene detection kit. Wherein,

[0026] The template DNA applicable to the kit of this invention is human genomic DNA. The DNA can be prepared from the following tissues or cells: blood (blood spots), semen (sperm spots), hair, saliva (saliva spots), sweat, amniotic fluid containing fetal cells, etc. A template DNA amount preferably in the range of 0.1 ng to 5 ng yields better amplification results. Too high a template amount will lead to nonspecific amplification results, while too low a template amount will result in some loci not being detected.

[0027] The amplification system of the kit uses polymerase chain reaction to amplify template DNA according to the specified reaction procedure, yielding a mixed amplification product of various loci. Because fluorescently labeled primers are used, the amplification products are also fluorescently labeled and their light signals can be identified using a genetic analyzer (such as ABI3130, ABI3500, etc.). The amplification products are mixed with a molecular weight internal standard and deionized formamide in a certain proportion, and then separated by electrophoresis using a capillary or gel in a genetic analyzer. The electrophoresis data can be analyzed on data analysis software such as GeneMapper and GeneMarker to obtain the genotyping map and data of the InDel locus.

[0028] The fluorescent multiplex amplification detection kit for the InDel site on human autosomes of the present invention can be used for human individual identification and paternity testing. Attached Figure Description

[0029] Figure 1 The InDel site typing map of sample 9947A.

[0030] Figure 2 The kit is used to genotype samples that have been ultrasonically broken down to simulate different degrees of degradation. Detailed Implementation

[0031] Although the principles of the present invention have been described in detail in the Summary of the Invention section, the technical content of the present invention will be further explained below in conjunction with specific embodiments in order to enable those skilled in the art to understand it more clearly.

[0032] Implementation Example 1

[0033] I. Determination of the InDel locus

[0034] By analyzing high-depth sequencing data from the 1000 Genome Project and other human genome genetic variation data from other genetic information databases, candidate InDel loci with high polymorphism and multiple alleles in East Asian populations were identified. The selected loci needed to meet the following criteria:

[0035] 1) Located on an autosome;

[0036] 2) They are distributed on different chromosomes, or located on the same chromosome with a distance between the two points greater than 10 Mb;

[0037] 3) The population contains at least three alleles of different lengths in East Asian populations, and the minimum frequency must be greater than 0.01;

[0038] 4) The locus satisfies Hardy-Weinberg equilibrium;

[0039] 5) Linkage equilibrium is maintained between gene loci.

[0040] Based on the InDel loci obtained through screening, polymorphism of each locus was evaluated and forensic parameters were calculated using 90 Han Chinese population samples. InDel loci with good amplification effects and high polymorphism were further screened. Based on the principle that the cumulative non-paternal exclusion probability is greater than 0.9999, 31 loci were selected to construct a detection system.

[0041] II. Construction of the Reagent Amplification System

[0042] When designing primers, repeated experiments are required to ensure that the primer length and amplified fragment size are appropriate for the testing of degraded samples (95-225bp), that the Tm values ​​are similar, and that primers do not form primer dimers. It is also necessary to ensure the specificity of primer amplification and to ensure that no non-specific products are generated. At the same time, the balance between various sites during multiplex amplification should be considered.

[0043] Based on extensive and in-depth research and experiments, this invention provides a fluorescent multiplex amplification kit containing 31 autosomal InDel loci and a sex identification gene. The PCR amplification system is shown in Table 2.

[0044] Table 2 Composition of the amplification system

[0045] 2×Multiplex PCR Master Mix 12.5μL 5× primer mixture 5μL Template DNA 0.1-5ng 4mg / ml BSA 1μL ddH2O Make up to 25μL

[0046] The primers and their concentrations for the 32 loci are shown in Table 1. The 2×Multiplex PCR MasterMix consisted of: 6 mM MgCl2, 100 mM Tris-HCl (pH = 8.7), 6 mM dNTPs, 100 mM KCl, and 0.4 U / μL hot-start Taq enzyme.

[0047] Implementation Example 2: Experimental method for detecting sample DNA using the fluorescent multiplex amplification kit from Example 1:

[0048] Using the fluorescence multiplex amplification kit in Application Example 1, genomic DNA extracted from 10 aliquots each of the 9947A genomic DNA standard (Promega, USA), bloodstains, semen (spots), and saliva (spots) was detected. 0.5 ng of genomic DNA was used as template DNA for each reaction. Multiplex PCR Master Mix, primer mixture, BSA, and ddH2O were prepared according to Table 2, and the mixture was vortexed and centrifuged in a PCR tube.

[0049] Set up the reaction cycler according to the following reaction conditions, place the PCR tubes inside, and begin the amplification reaction. Incubate at 95°C for 5 minutes; incubate at 94°C for 30 seconds, at 60°C for 90 seconds, at 70°C for 30 seconds, and repeat this cycle for 29 times; incubate at 60°C for 35 minutes; incubate at 4°C until the samples are removed.

[0050] The amplification products were detected and analyzed using a genetic analyzer.

[0051] The total loading premix for each sample was calculated using 3.85 μL of deionized formamide and 0.15 μL of the molecular weight internal standard (500 LIZ standard). 4 μL of the loading premix was mixed with 1 μL of the amplification product from this kit, centrifuged, denatured at 95°C for 5 minutes, and then incubated on ice for 5 minutes. Detection was performed using a genetic analyzer, and the data collected by the genetic analyzer was analyzed using GeneMapper.

[0052] Experimental results: The fluorescent multiplex amplification kit has good results in InDel typing of DNA samples. Figure 1 The InDel typing results for the 9947A genomic DNA standard are shown.

[0053] Implementation Example 3

[0054] The fluorescent multiplex amplification kit in Application Example 1 is used to detect DNA in simulated degradation biological samples by ultrasonic fragmentation.

[0055] Experimental methods:

[0056] Peripheral blood genomic DNA was prepared using the QIAamp DNA Investigator Kit (QIAGEN). Five genomic DNA samples, 200 ng each, were prepared and fragmented into samples with average fragment sizes of <150 bp, 150 bp, 200 bp, 300 bp, and 400 bp using a Covaris S220 Focused-ultrasonicator. Multiplex PCR Master Mix, primer mixture, BSA, and ddH2O were prepared according to Table 2. 0.5 ng of each fragmented DNA sample was added to a PCR tube and vortexed to mix.

[0057] Set up the reaction cycler according to the following reaction conditions, and place the PCR tubes inside for amplification. Incubate at 95℃ for 5 minutes; incubate at 94℃ for 30 seconds, at 60℃ for 90 seconds, at 70℃ for 30 seconds, and repeat this cycle for 29 times; incubate at 60℃ for 35 minutes; incubate at 4℃ until the samples are removed.

[0058] The amplification products were detected and analyzed using a genetic analyzer.

[0059] The total loading premix for each sample was calculated using 3.85 μL of deionized formamide and 0.15 μL of the molecular weight internal standard (500 LIZ standard). 4 μL of the loading premix was mixed with 1 μL of the amplification product from this kit, centrifuged, denatured at 95°C for 5 minutes, and then incubated on ice for 5 minutes. Detection was performed using a genetic analyzer, and the data collected by the genetic analyzer was analyzed using GeneMapper.

[0060] Experimental results: Figure 2 The InDel typing results of the kit for simulated degradation samples with different degrees of ultrasonic fragmentation are shown. The results show that for degradation samples with an average fragment length of less than 150 bp, only one locus was not detected by the kit, and the detection rate of the rest was 100%. This indicates that the present invention has shown good detection effect in the detection of degradation samples due to the shorter amplified fragment.

[0061] The embodiments described are not intended to limit the scope of protection of this invention, but rather to disclose the technical content of this invention in more detail. Any modifications and / or changes that are obvious to those skilled in the art will also be included in the concept of this invention. sequence list <110> Fudan University <120> A fluorescent multiplex amplification detection kit for the InDel site on human autosomes and its application <160> 64 <170> SIPOSequenceList 1.0 <210> 1 <211> 20 <212> DNA <213> HID1-1 Forward first <400> 1 ttagctgga gagtggtagc <210> 2 <211> 20 <212> DNA <213> HID1-1 Reverse primer <400> 2 ggcaatccgg acagacttat <210> 3 <211> 22 <212> DNA <213> HID1-2 Forward first <400> 3 catcgttgag gggaagagta tc <210> 4 <211> 23 <212> DNA <213> HID1-2 Reverse primer <400> 4 23. snow snow snow <210> 5 <211> 18 <212> DNA <213> HID1-3 Forward first <400> 5 cttcttac 18. cttcttac <210> 6 <211> 20 <212> DNA <213> HID1-3 Reverse primer <400> 6 tgtccttggg atagtaagtt 20 <210> 7 <211> 25 <212> DNA <213> HID1-4 Forward primer <400> 7 ttttgccatt tgataggttt atgag 25 <210> 8 <211> 21 <212> DNA <213> HID1-4 Reverse primer <400> 8 agacagccca agagatcaat g 21 <210> 9 <211> 18 <212> DNA <213> HID2-1 Forward primer <400> 9 gggtttaagg gagtgatt 18 <210> 10 <211> 20 <212> DNA <213> HID2-1 Reverse primer <400> 10 gttcattgct atcaatatgc 20 <210> 11 <211> 23 <212> DNA <213> HID3-1 Forward primer <400> 11 cacgggaatt cagagaaaag aat 23 <210> 12 <211> 20 <212> DNA <213> HID3-1 Reverse primer <400> 12 ggcaccttct ttaggatgac 20 <210> 13 <211> 22 <212> DNA <213> HID3-2 Forward primer <400> 13 ggtccctcct tcaatgtatc cc 22 <210> 14 <211> 22 <212> DNA <213> HID3-2 Reverse primer <400> 14 ggtagtgggt aatgaggatg cc 22 <210> 15 <211> 22 <212> DNA <213> HID4-1 Forward primer <400> 15 tgaaggattt ggtaggtgat gt 22 <210> 16 <211> 20 <212> DNA <213> HID4-1 Reverse primer <400> 16 actccaaagg accagttcac 20 <210> 17 <211> 22 <212> DNA <213> HID4-2 Forward primer <400> 17 ttccccagtg taagacacac ag 22 <210> 18 <211> 22 <212> DNA <213> HID4-2 Reverse primer <400> 18 aaatcctgag tgtgcctctg at 22 <210> 19 <211> 25 <212> DNA <213> HID5-1 Forward primer <400> 19 ctgtgctgag aaggatctta gaaac 25 <210> 20 <211> 23 <212> DNA <213> HID5-1 Reverse primer <400> 20 tctgtgatgc cgtattctgt gac 23 <210> 21 <211> 25 <212> DNA <213> HID6-1 Forward primer <400> 21 gtaaaggttt ttcaacttat cagga 25 <210> 22 <211> 25 <212> DNA <213> HID6-1 Reverse primer <400> 22 tctgagcaaa tacttagtat tctgc 25 <210> 23 <211> 19 <212> DNA <213> HID6-2 Forward primer <400> 23 tttggtcagt ccaggggtc 19 <210> 24 <211> 24 <212> DNA <213> HID6-2 Reverse primer <400> 24 taatcagaag tgtgggtaca aatg 24 <210> 25 <211> 24 <212> DNA <213> HID7-1 Forward primer <400> 25 gcaaaataag tgacttcaca atgg 24 <210> 26 <211> 22 <212> DNA <213> HID7-1 Reverse primer <400> 26 tctgcaatgc aaaaacactg ag 22 <210> 27 <211> 22 <212> DNA <213> HID7-2 Forward primer <400> 27 gcagttatgc ttggaattct ct 22 <210> 28 <211> 24 <212> DNA <213> HID7-2 Reverse primer <400> 28 tctaaattgt gtttctttgt ggtc 24 <210> 29 <211> 23 <212> DNA <213> HID7-3 Forward primer <400> 29 ccccttagag ccatattttc tga 23 <210> 30 <211> 22 <212> DNA <213> HID7-3 Reverse primer <400> 30 tgccagagca acagtatcct ac 22 <210> 31 <211> 22 <212> DNA <213> HID8-1 Forward primer <400> 31 gttcaggggt aaagtgtcat gg <210> 32 <211> 25 <212> DNA <213> HID8-1 Reverse primer <400> 32 aagtggactg aatttcttct tcaat <210> 33 <211> 20 <212> DNA <213> HID8-2 Forward primer <400> 33 cagcacctca atgaatgtgt <210> 34 <211> 24 <212> DNA <213> HID8-2 Reverse primer <400> 34 fatheraaaa gcagggttgc ttgg 24 <210> 35 <211> 24 <212> DNA <213> HID9-1 Forward primer <400> 35 tttcaagttg atgatgtacc tctg <210> 36 <211> 21 <212> DNA <213> HID9-1 Reverse primer <400> 36 gtaacaagcc gagttagact g <210> 37 <211> 20 <212> DNA <213> HID9-2 Forward primer <400> 37 cgttgatgta gtggagggag <210> 38 <211> 20 <212> DNA <213> HID9-2 Reverse primer <400> 38 cagttcagca ccttggacag <210> 39 <211> 23 <212> DNA <213> HID11-1 Forward primer <400> 39 tttgtagggg tcagattagc tgc <210> 40 <211> 24 <212> DNA <213> HID11-1 Reverse primer <400> 40 ggcctgtgga aaatccagat aaag <210> 41 <211> 19 <212> DNA <213> HID11-2 Forward primer <400> 41 gtcctgccct aggtgtgag <210> 42 <211> 22 <212> DNA <213> HID11-2 Reverse primer <400> 42 catggtccat caactcattc ac <210> 43 <211> 20 <212> DNA <213> HID11-3 Forward primer <400> 43 aatgtaaggt ggtgtcaggt 20 <210> 44 <211> 20 <212> DNA <213> HID11-3 Reverse primer <400> 44 taggggctaa gctttgtctg 20 <210> 45 <211> 22 <212> DNA <213> HID12-1 Forward primer <400> 45 gagaagcagc tagcacctct tg 22 <210> 46 <211> 22 <212> DNA <213> HID12-1 Reverse primer <400> 46 aagcagaagg ggttatgtga gc 22 <210> 47 <211> 22 <212> DNA <213> HID13-1 Forward primer <400> 47 catgggagta ggacaaggta ac 22 <210> 48 <211> 26 <212> DNA <213> HID13-1 Reverse primer <400> 48 catttacaat agaacaaccc tatctt 26 <210> 49 <211> 24 <212> DNA <213> HID14-1 Forward primer <400> 49 tgtcaatgaa attttgagtt cagg 24 <210> 50 <211> 22 <212> DNA <213> HID14-1 Reverse primer <400> 50 ggcatgaaaa gcacaacaca ag 22 <210> 51 <211> 22 <212> DNA <213> HID14-2 Forward primer <400> 51 ggccatcagt gggagacttc tg 22 <210> 52 <211> 22 <212> DNA <213> HID14-2 Reverse primer <400> 52 ccagtactcg gaggataggt cc 22 <210> 53 <211> 23 <212> DNA <213> HID16-1 Forward primer <400> 53 aacagtaaga tggtggtcag gag 23 <210> 54 <211> 23 <212> DNA <213> HID16-1 Reverse primer <400> 54 tagggatggg agttaaaggg acc 23 <210> 55 <211> 25 <212> DNA <213> HID18-1 Forward primer <400> 55 aagtgttact gtcatctcaa gcctc 25 <210> 56 <211> 25 <212> DNA <213> HID18-1 Reverse primer <400> 56 caatggagaa gaaagtggct gatag 25 <210> 57 <211> 19 <212> DNA <213> HID19-1 Forward primer <400> 57 accatgccca tccgagact 19 <210> 58 <211> 22 <212> DNA <213> HID19-1 Reverse primer <400> 58 aaattctcac aagtgcacaa gg 22 <210> 59 <211> 24 <212> DNA <213> HID20-1 Forward primer <400> 59 ggccagatga tttcttatat ccat 24 <210> 60 <211> 22 <212> DNA <213> HID20-1 Reverse primer <400> 60 gcattaacat acccaaacac ac 22 <210> 61 <211> 19 <212> DNA <213> HID22-1 Forward primer <400> 61 caagcctacc ctacctccg 19 <210> 62 <211> 24 <212> DNA <213> HID22-1 Reverse primer <400> 62 ggttctgaat tcatctaatg gctg 24 <210> 63 <211> 23 <212> DNA <213> Amelogenin Forward primer <400> 63 cctgggctct gtaaagaata gtg 23 <210> 64 <211> 22 <212> DNA <213> Amelogenin Reverse primer <400> 64 cagagcttaa actgggaagc tg 22

Claims

1. A gene detection kit, characterized in that, This kit contains 31 pairs of primers specifically for amplifying autosomal InDel loci, and one pair of primers specifically for amplifying sex-identifying genes. The autosomal InDel loci are as follows: HID1-1: rs60893206, HID1-2: rs58568060, HID1-3: rs1553161226, HID1-4: rs34314833, HID2-1: rs77948797, HID3-1: rs139809929, HID3-2: rs38667001 7. HID4-1: rs3077701, HID4-2: rs70949989, HID5-1: rs200753977, HID6-1: rs10635385, HID6-2: rs1554219917, HID7 -1: rs58558819, HID7-2: rs6150152, HID7-3: rs112257258, HID8-1: rs10596541, HID8-2: rs59118283, HID9-1: rs386 737038, HID9-2: rs138815275, HID11-1: rs11271121, HID11-2: rs66572312, HID11-3: rs1555121221, HID12-1: rs614 4790, HID13-1: rs386768785, HID14-1: rs33958617, HID14-2: rs58233606, HID16-1: rs1383237378, HID18-1: rs7721 91138, HID19-1: rs113712314, HID20-1: rs11467514, HID22-1: rs758153887, where rs is the locus number in the dbSNP database; the sex identification gene is the Amelogenin gene; the autosomal InDel locus contains at least 3 alleles in the population; the PCR amplification products of the specific amplification primers are all less than 225 bp in length; the specific amplification primer sequences are selected from the following sequences: HID1-1, SEQ HID1-2, SEQ ID NO.3-4; HID1-3, SEQ ID NO.5-6; HID1-4, SEQ ID NO.7-8; HID2-1, SEQ ID NO.9-10; HID3-1, SEQ ID NO.11-12; HID3-2, SEQ ID NO.13-14; HID4-1, SEQ ID NO. NO.15-16; HID4-2, SEQ ID NO.17-18; HID5-1, SEQ ID NO.19-20;HID6-1,SEQ ID NO.21-22;HID6-2,SEQ IDNO.23-24;HID7-1,SEQ ID NO.25-26;HID7-2,SEQ ID NO.27-28;HID7-3,SEQ ID NO.29-30;HID8-1,SEQ ID NO.31-32;HID8-2,SEQ ID NO.33-34;HID9-1,SEQ ID NO.35-36;HID9-2,SEQ ID NO.37-38;HID11-1,SEQ ID NO.39-40;HID11-2,SEQ ID NO.41-42;HID11-3,SEQID NO.43-44;HID12-1,SEQ ID NO.45-46;HID13-1,SEQ ID NO.47-48;HID14-1,SEQ IDNO.49-50;HID14-2,SEQ ID NO.51-52;HID16-1,SEQ ID NO.53-54;HID18-1,SEQ IDNO.55-56;HID19-1,SEQ ID NO.57-58;HID20-1,SEQ ID NO.59-60;HID22-1,SEQ IDNO.61-62;Amelogenin,SEQ ID NO.63-64。.

2. The gene detection kit according to claim 1, characterized in that, The specific amplification primers were prepared at the following concentrations: HID1-1, 0.48 μM; HID1-2, 0.48 μM; HID1-3, 0.48 μM; HID1-4, 0.48 μM; HID2-1, 0.48 μM; HID3-1, 0.48 μM; HID3-2, 0.08 μM; HID4-1, 0.48 μM; HID4-2, 0.48 μM. HID5-1, 0.48μM; HID6-1, 0.48μM; HID6-2, 0.12μM; HID7-1, 0.24μM; HID7-2, 0.12μM; HID7-3, 0.24μM; HID8-1, 0.16μM; HID8-2, 0.24μM; HID9-1, 0.12μM; HID9-2, 0.48μM; HID11-1, 0.12μM; HID11-2, 0.48μM ;HID11-3, 0.48μM; HID12-1, 0.16μM; HID13-1, 0.48μM; HID14-1, 0.16μM; HID14-2, 0.16μM; HID16-1, 0.16 μM; HID18-1, 0.08 μM; HID19-1, 0.12 μM; HID20-1, 0.12 μM; HID22-1, 0.96 μM; Amelogenin, 0.48 μM.

3. The gene detection kit according to claim 1, characterized in that, The specific amplification products were divided into four groups, as follows: Group 1 consisted of HID3-2, HID7-1, HID8-1, HID12-1, HID14-1, HID14-2, HID16-1, and HID18-1; Group 2 consisted of HID6-2, HID7-2, HID7-3, HID8-2, HID9-1, HID11-1, HID19-1, and HID20-1; Group 3 consisted of HID1-1, HID1-4, and HID20-1. The primers in the first group are HID1, HID3-1, HID9-2, HID11-3, and HID22-1; the fourth group is Amelogenin, HID1-2, HID1-3, HID4-1, HID4-2, HID5-1, HID6-1, HID11-2, and HID13-1. In each primer pair, the 5′ end of one primer is labeled with a fluorescent dye. The four groups of primers are labeled with blue, green, yellow, and red fluorescent dyes, respectively, and the fluorescent label used in each group is different.

4. The gene detection kit according to claim 3, characterized in that, The primers were labeled using the following methods: blue labels were made using 5-FAM (5-carboxyfluorescein), 6-FAM (6-carboxyfluorescein), or fluorescein molecules with similar spectra; green labels were made using HEX (hexachloro-6-methylfluorescein), JOE (6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein succinimide), or fluorescein molecules with similar spectra; yellow labels were made using TAMRA (carboxytetramethylrhodamine), or fluorescein molecules with similar spectra; and red labels were made using ROX (carboxy-X-rhodamine), or fluorescein molecules with similar spectra.

5. The gene detection kit according to claim 3, characterized in that, The specific amplification primers and their fluorescent labels are as follows: The fluorescent dye molecules used to label the primers are 6-FAM, HEX, TAMRA, and ROX; specifically: 6-FAM labels HID3-2, HID7-1, HID8-1, HID12-1, HID14-1, HID14-2, HID16-1, and HID18-1; HEX labels HID6-2, HID7-2, HID7-3, HID8-2, and H... ID9-1, HID11-1, HID19-1, HID20-1; TAMRA-labeled HID1-1, HID1-4, HID2-1, HID3-1, HID9-2, HID11-3, HID22-1; ROX-labeled Amelogenin, HID1-2, HID1-3, HID4-1, HID4-2, HID5-1, HID6-1, HID11-2, HID13-1.

6. The gene detection kit according to claim 1, characterized in that, The kit contains 2×Multiplex PCR Master Mix; 5×Primer Mixture; BSA; Hot-start Taq Enzyme; ddH2O.

7. The use of the gene detection kit according to any one of claims 1-6 in the preparation of products for human individual identification and paternity testing.

8. The application according to claim 7, characterized in that, The application uses polymerase chain reaction to amplify the InDel locus and sex identification site, and then performs typing by detecting the products using capillary electrophoresis.

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