A kit and detection method for blood source identification and personal identification based on CpG-SNP linkage markers

Abstract

The present invention belongs to the technical field of forensic detection, and specifically relates to a kit and a detection method for blood source identification and personal identification based on CpG-SNP linkage markers, wherein the kit comprises 23 pairs of specific multiplex PCR primers shown in SEQ ID NO.1-46, 22 CpG single base extension primers shown in SEQ ID NO.47-68, and 24 SNP single base extension primers shown in SEQ ID NO.69-92. Through bisulfite conversion, composite amplification, SNaPshot single base extension reaction, capillary electrophoresis and result analysis, the blood components in the mixed spots are detected and the blood donors are individually typed, and the blood and its donors are linked, which can be used for forensic identification of mixed spots containing blood. The technology is simple and easy to operate, has low cost, and is easy to popularize at the grassroots level.

Description

Technical Field

[0001] The present invention belongs to the technical field of forensic detection, and specifically relates to a kit and a detection method for blood source identification and individual identification based on CpG-SNP linkage markers, and more particularly to a kit and a detection method for simultaneously identifying the body fluid source and individual identification of blood components in mixed spots based on body fluid-specific differential methylation sites combined with one or more SNP linkage markers. Background Art

[0002] There are many traditional methods for identifying bloodstains, such as benzidine and luminol tests. However, these tests can only analyze a single body fluid and may produce false positives due to the influence of other body fluids in mixed spots, and hinder downstream applications such as DNA analysis. In recent years, tissue-specific mRNA has also been reported to be a molecular marker for distinguishing body fluids. It has high specificity, can simultaneously analyze multiple components in mixed spots, and can be co-extracted with DNA. Unfortunately, biological materials in forensic investigations are often exposed to harsh conditions for a long time, and mRNA is extremely unstable, which limits its application in daily forensic work. In addition, there are microbial methods based on body fluid specificity, but they are currently limited to detecting vaginal secretions and saliva. Based on the fact that DNA is more stable than RNA, DNA methylation is considered a promising tool for distinguishing various body fluids. As an epigenetic modification, DNA methylation usually occurs at the 5' position of the cytosine pyrimidine ring in CpG dinucleotides and plays an important role in regulating gene expression. Many studies have found that DNA methylation sites (i.e., CpG sites) in different tissues or body fluids exhibit different methylation patterns and have been successfully validated for the identification of body fluids.

[0003] However, most studies only use DNA methylation to identify the source of body fluids in mixed spots, but the identification of personal identity in the case is more meaningful and valuable. At present, STR typing is a routine method in the analysis of DNA mixtures, but its accuracy is interfered by many factors, such as allele sharing (masking effect), stutter peaks, allele loss or insertion, etc. In addition, extremely uneven mixtures are often obtained at crime scenes, but the detection limit of STR typing is based on minor contributors accounting for at least 5%-10% of the total mixture. Therefore, in the process of analyzing unbalanced DNA mixtures, some new linkage markers have gradually emerged, such as SNP-STR, DIP-SNP, and DIP-microhaplotype, but like STR, they cannot identify the source of body fluids.

[0004] In recent years, many studies have shown that there are SNP sites near CpG sites, and CpG-linked SNPs can be used to identify individuals while determining the source of body fluids in mixed spots. Therefore, this method uses such linked markers, first using CpG sites for body fluid-specific amplification, and then using the polymorphism of SNP sites to identify individuals, which is a more complete solution. Summary of the invention

[0005] The purpose of the present invention is to simultaneously identify the source of blood components in mixed spots and perform individual typing based on body fluid-specific differential methylation sites combined with one or more SNP-linked markers, thereby linking body fluids with donors, thereby providing a new idea for the analysis of mixed spots.

[0006] The present invention is achieved through the following technical scheme: a kit for blood source identification and personal identification based on CpG-SNP linkage markers, comprising 23 pairs of specific multiplex PCR primers as shown in SEQ ID NO.1-46, for detecting the following 23 markers, namely, specifically detecting 19 blood-specific CpG-SNP linkage markers, 1 semen-specific CpG site, 1 saliva-specific CpG site, 1 vaginal secretion-specific CpG site, and 1 internal control, i.e., housekeeping gene, for evaluating bisulfite conversion efficiency ACTB .

[0007] Among them, 19 blood-specific CpG-SNP linkage markers were cg19182633-rs2072110, cg01022219-rs4796991, cg04011671-rs4640436-rs72876044, cg06442294-rs10419232, cg11239720-rs58781231, cg01607849-rs12302749, cg19987129-rs6925308, cg09234599-rs11609960-rs10846771-rs7977743, and cg2412443 -rs68094061, cg03818715-rs2744388, cg15033269-rs587712, cg06645778-rs55988613, cg13581015-rs10848543, cg00578614-rs2523994, c g03666441-rs66687178, cg23927970-rs11827672, cg12237948-rs6132289, cg19092837-rs35111552-rs11121529, cg08792630-rs58775446;

[0008] One semen-specific CpG site is cg13885748;

[0009] One saliva-specific CpG site is cg26107890;

[0010] One vaginal secretion-specific CpG site was cg09765089.

[0011] The present invention selects 19 blood-specific sites and screens SNP molecular markers with a minimum allele frequency (MAF) ≥ 0.1 within 400 bp upstream and downstream to form CpG-SNP linkage markers. Furthermore, the present invention also additionally screens 1 semen-specific CpG site, 1 saliva-specific CpG site, and 1 vaginal secretion-specific CpG site for the identification of these three body fluids in mixed spots, as well as an internal control for evaluating the efficiency of bisulfite conversion, i.e., a housekeeping gene ACTB .

[0012] The relevant information of the above 23 markers is shown in Table 1.

[0013] Table 1 Relevant information of 23 markers

[0014]

[0015] As a further improvement of the technical solution of the present invention, the specific PCR primers for cg19182633-rs2072110 are the upstream primer of SEQ ID NO.1 and the downstream primer of SEQ ID NO.2;

[0016] The specific PCR primers for cg01022219-rs4796991 are the upstream primer of SEQ ID NO.3 and the downstream primer of SEQ ID NO.4;

[0017] The specific PCR primers for cg04011671-rs4640436-rs72876044 are the upstream primer of SEQ ID NO.5 and the downstream primer of SEQ ID NO.6;

[0018] The specific PCR primers for cg06442294-rs10419232 are the upstream primer of SEQ ID NO.7 and the downstream primer of SEQ ID NO.8;

[0019] The specific PCR primers for cg11239720-rs58781231 are the upstream primer of SEQ ID NO.9 and the downstream primer of SEQ ID NO.10;

[0020] The specific PCR primers for cg01607849-rs12302749 are the upstream primer of SEQ ID NO.11 and the downstream primer of SEQ ID NO.12;

[0021] The specific PCR primers for cg19987129-rs6925308 are the upstream primer of SEQ ID NO.13 and the downstream primer of SEQ ID NO.14;

[0022] The specific PCR primers for cg09234599-rs11609960-rs10846771-rs7977743 are the upstream primer of SEQ ID NO.15 and the downstream primer of SEQ ID NO.16;

[0023] The specific PCR primers for cg24124443-rs68094061 are the upstream primer of SEQ ID NO.17 and the downstream primer of SEQ ID NO.18;

[0024] The specific PCR primers for cg03818715-rs2744388 are the upstream primer of SEQ ID NO.19 and the downstream primer of SEQ ID NO.20;

[0025] The specific PCR primers for cg15033269-rs587712 are the upstream primer of SEQ ID NO.21 and the downstream primer of SEQ ID NO.22;

[0026] The specific PCR primers for cg06645778-rs55988613 are the upstream primer of SEQ ID NO.23 and the downstream primer of SEQ ID NO.24;

[0027] The specific PCR primers for cg13581015-rs10848543 are the upstream primer of SEQ ID NO.25 and the downstream primer of SEQ ID NO.26;

[0028] The specific PCR primers for cg00578614-rs2523994 are the upstream primer of SEQ ID NO.27 and the downstream primer of SEQ ID NO.28;

[0029] The specific PCR primers for cg03666441-rs66687178 are the upstream primer of SEQ ID NO.29 and the downstream primer of SEQ ID NO.30;

[0030] The specific PCR primers for cg23927970-rs11827672 are the upstream primer of SEQ ID NO.31 and the downstream primer of SEQ ID NO.32;

[0031] The specific PCR primers for cg12237948-rs6132289 are the upstream primer of SEQ ID NO.33 and the downstream primer of SEQ ID NO.34;

[0032] The specific PCR primers for cg19092837-rs35111552-rs11121529 are the upstream primer of SEQ ID NO.35 and the downstream primer of SEQ ID NO.36;

[0033] The specific PCR primers for cg08792630-rs58775446 are the upstream primer of SEQ ID NO.37 and the downstream primer of SEQ ID NO.38;

[0034] The specific PCR primers for cg13885748 are the upstream primer of SEQ ID NO.39 and the downstream primer of SEQ ID NO.40;

[0035] The specific PCR primers for cg26107890 are the upstream primer of SEQ ID NO.41 and the downstream primer of SEQ ID NO.42;

[0036] The specific PCR primers for cg09765089 are the upstream primer of SEQ ID NO. 43 and the downstream primer of SEQ ID NO 44;

[0037] ACTB The specific PCR primers are the upstream primer of SEQ ID NO.45 and the downstream primer of SEQ ID NO.46.

[0038] The 19 blood-specific CpG-SNP linkage markers, 1 semen-specific CpG site, 1 saliva-specific CpG site, 1 vaginal secretion-specific CpG site and a housekeeping gene of the present invention ACTB The nucleotide sequences of the upstream and downstream primers are shown in Table 2.

[0039] Table 2 Nucleotide sequences of primers used for amplification and detection of markers

[0040]

[0041]

[0042] Note: Underlines in primer sequences indicate tissue-specific sites, and lowercase letters indicate introduced mismatched bases.

[0043] As a further improvement of the technical solution of the present invention, it also includes 22 CpG single base extension primers as shown in SEQ ID NOs. 47 to 68, corresponding to 19 blood-specific CpG-SNP linkage markers, 1 semen-specific CpG site, 1 saliva-specific CpG site, and 1 vaginal secretion-specific CpG site;

[0044] Wherein: the extension primer sequence corresponding to cg19182633 is shown in SEQ ID NO.47;

[0045] The extension primer sequence corresponding to cg01022219 is shown in SEQ ID NO.48;

[0046] The extension primer sequence corresponding to cg04011671 is shown in SEQ ID NO.49;

[0047] The extension primer sequence corresponding to cg06442294 is shown in SEQ ID NO.50;

[0048] The extension primer sequence corresponding to cg11239720 is shown in SEQ ID NO.51;

[0049] The extension primer sequence corresponding to cg01607849 is shown in SEQ ID NO.52;

[0050] The extension primer sequence corresponding to cg19987129 is shown in SEQ ID NO.53;

[0051] The extension primer sequence corresponding to cg09234599 is shown in SEQ ID NO.54;

[0052] The extension primer sequence corresponding to cg24124443 is shown in SEQ ID NO.55;

[0053] The extension primer sequence corresponding to cg03818715 is shown in SEQ ID NO.56;

[0054] The extension primer sequence corresponding to cg15033269 is shown in SEQ ID NO.57;

[0055] The extension primer sequence corresponding to cg06645778 is shown in SEQ ID NO.58;

[0056] The extension primer sequence corresponding to cg13581015 is shown in SEQ ID NO.59;

[0057] The extension primer sequence corresponding to cg00578614 is shown in SEQ ID NO.60;

[0058] The extension primer sequence corresponding to cg03666441 is shown in SEQ ID NO.61;

[0059] The extension primer sequence corresponding to cg23927970 is shown in SEQ ID NO.62;

[0060] The extension primer sequence corresponding to cg12237948 is shown in SEQ ID NO.63;

[0061] The extension primer sequence corresponding to cg19092837 is shown in SEQ ID NO.64;

[0062] The extension primer sequence corresponding to cg08792630 is shown in SEQ ID NO.65;

[0063] The extension primer sequence corresponding to cg13885748 is shown in SEQ ID NO.66;

[0064] The extension primer sequence corresponding to cg26107890 is shown in SEQ ID NO.67;

[0065] The extension primer sequence corresponding to cg09765089 is shown in SEQ ID NO.68.

[0066] Specifically, the information of 22 CpG single base extension primers is shown in Table 3:

[0067] Table 3 22 CpG single base extension primers

[0068]

[0069] As a further improvement of the technical solution of the present invention, 24 SNP single base extension primers as shown in SEQ ID NOs. 69 to 92 are also included, which correspond to 23 SNP molecular markers and housekeeping genes linked to 19 blood-specific CpG sites. ACTB ;

[0070] The extension primer sequence corresponding to rs2072110 is as SEQ ID NO.69;

[0071] The extension primer sequence corresponding to rs4796991 is as SEQ ID NO.70;

[0072] The extension primer sequence corresponding to rs4640436 is as SEQ ID NO.71;

[0073] The extension primer sequence corresponding to rs72876044 is as SEQ ID NO.72;

[0074] The extension primer sequence corresponding to rs10419232 is as SEQ ID NO.73;

[0075] The extension primer sequence corresponding to rs58781231 is as SEQ ID NO.74;

[0076] The extension primer sequence corresponding to rs12302749 is as SEQ ID NO.75;

[0077] The extension primer sequence corresponding to rs6925308 is as SEQ ID NO.76;

[0078] The extension primer sequence corresponding to rs11609960 is as SEQ ID NO.77;

[0079] The extension primer sequence corresponding to rs10846771 is as SEQ ID NO.78;

[0080] The extension primer sequence corresponding to rs7977743 is as SEQ ID NO.79;

[0081] The extension primer sequence corresponding to rs68094061 is as SEQ ID NO.80;

[0082] The extension primer sequence corresponding to rs2744388 is as SEQ ID NO.81;

[0083] The extension primer sequence corresponding to rs587712 is as SEQ ID NO.82;

[0084] The extension primer sequence corresponding to rs55988613 is as SEQ ID NO.83;

[0085] The extension primer sequence corresponding to rs10848543 is as SEQ ID NO.84;

[0086] The extension primer sequence corresponding to rs2523994 is as SEQ ID NO.85;

[0087] The extension primer sequence corresponding to rs66687178 is as SEQ ID NO.86;

[0088] The extension primer sequence corresponding to rs11827672 is as SEQ ID NO.87;

[0089] The extension primer sequence corresponding to rs6132289 is as SEQ ID NO.88;

[0090] The extension primer sequence corresponding to rs35111552 is as SEQ ID NO.89;

[0091] The extension primer sequence corresponding to rs11121529 is as SEQ ID NO.90;

[0092] The extension primer sequence corresponding to rs58775446 is as SEQ ID NO.91;

[0093] ACTB The corresponding extension primer sequence is shown as SEQ ID NO.92.

[0094] The information of 24 SNP single base extension primers is shown in Table 4:

[0095] Table 4 24 SNP single base extension primers

[0096]

[0097] The kit for blood source identification and personal identification based on CpG-SNP linkage markers provided by the present invention uses body fluid-specific differential methylation sites to infer the sources of blood, semen, saliva, and vaginal secretions in mixed spots, and simultaneously uses SNP molecular markers linked to blood-specific methylation sites to achieve personal identification of blood donors in mixed spots.

[0098] As a further improvement of the technical solution of the present invention, each pair of specific multiplex PCR primers contains at least one blood-specific CpG site-linked SNP and at most three.

[0099] As a further improvement of the technical solution of the present invention, the kit also includes deionized formamide and molecular weight internal standard GeneScan-120 LIZ for detecting single base extension products.

[0100] The present invention further provides a method for blood source identification and individual identification based on CpG-SNP linkage markers, comprising the following steps:

[0101] 1) extracting the DNA of the sample to be tested as a template, treating it with bisulfite, and using the 23 pairs of specific multiplex PCR primers shown in SEQ ID NOs. 1 to 46 to obtain amplification products by multiplex specific PCR;

[0102] 2) Purifying the amplified product, and obtaining the extension product by a single base extension system using 22 CpG single base extension primers as shown in SEQ ID NOs. 47 to 68 and 24 SNP single base extension primers as shown in SEQ ID NOs. 69 to 92;

[0103] 3) The extended products are purified and analyzed using capillary electrophoresis, 3500 sequencer and gene analysis software to complete blood source identification and personal identification.

[0104] Compared with the prior art, the present invention has the following advantages:

[0105] (1) This kit is based on a capillary electrophoresis platform, which is commonly used by grassroots forensic scientists. It also uses SNaPshot technology, which is simple, easy to use, and low-cost, making it easy to popularize at the grassroots level.

[0106] (2) This detection method can detect 19 blood-specific CpG markers and three other body fluid CpG indicator markers at one time. These CpG sites have good tissue specificity and there is no interference from amplification product peaks in other non-target body fluids, so the source of body fluids can be accurately identified.

[0107] (3) While identifying body fluids, individual typing can be performed. The 23 SNPs linked to the blood-specific CpG markers have good polymorphism and high cumulative individual recognition ability, making them suitable for forensic identification and testing.

[0108] (4) Through bisulfite conversion, composite amplification, SNaPshot single base extension reaction, capillary electrophoresis and result analysis, the blood components in the mixed spots are detected and the blood donors are individually typed, and the blood and its donor are linked, which can be used for forensic identification of mixed spots containing blood. It can be used to simultaneously analyze the body fluid source of the sample and personal identification, providing a new idea for forensic identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0110] Figure 1 The tissue-specific maps are 19 blood-specific CpG sites and 1 CpG site in semen, saliva, and vaginal secretions. As can be seen from the figure: 19 blood-specific CpG markers were successfully detected in blood samples, but not in the other three body fluids. Similarly, the three indicator markers showed good specificity in the multiplex detection system, so this system can accurately identify the presence of these four body fluids.

[0111] Figure 2This is an electrophoresis analysis of a random individual in a population frequency survey. It can be seen from the figure that the internal control ACTB was successfully amplified, indicating that the sample was fully converted. At the same time, 23 SNP markers were successfully typed in the blood sample, indicating that the system can accurately identify individual blood donors.

[0112] Figure 3 This is the CpG map of a blood spot sample stored at room temperature for 9 months. In the figure, letters a, b, c, d and e represent the test results of blood samples placed at room temperature for 0 days (fresh sample), 2 months, 5 months, 7 months and 9 months respectively. It can be seen from the figure that blood-specific methylation markers are not affected by storage time and remain stable over a long period of time, so they can be reliably used for blood identification.

[0113] Figure 4 This is the SNP typing map of a blood spot sample stored at room temperature for 9 months. In the figure, letters a, b, c, d and e represent the test results of blood samples stored at room temperature for 0 days (fresh sample), 2 months, 5 months, 7 months and 9 months respectively. It can be seen from the figure that blood-specific SNP polymorphism markers, like CpG markers, remain stable under long-term storage conditions, that is, SNP typing is not affected and can be accurately used for personal identification of blood donors. DETAILED DESCRIPTION

[0114] The technical solution of the present invention is described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0115] Example 1: Construction of a multiplex linked marker detection system for identifying the blood source in mixed plaques and identifying individual blood donors.

[0116] 1. Screening of specific CpG-SNP linked markers:

[0117] 1) Through literature screening, candidate blood-specific CpG sites and one specific CpG site for each of the other three body fluids in the mixed plaque were obtained, namely, one semen-specific CpG site, one saliva-specific CpG site, and one vaginal secretion-specific CpG site.

[0118] 2) Screen SNP molecular markers with MAF values ​​≥ 0.1 within 400 bp upstream and downstream of the blood-specific CpG site.

[0119] 2. Design primers for linked markers: the target fragment length does not exceed 400bp, the primer length does not exceed 35bp, the 3' end of the primer is completely complementary to the CpG site of the target body fluid, and an artificial mismatch is introduced at the second to last or third position of the 3' end to improve the efficiency of specific amplification.

[0120] 3. Tissue specificity verification: The candidate CpG sites of the four body fluids were preliminarily verified by methylation-specific PCR. Specifically, each site was amplified separately in the four body fluid samples, and its tissue specificity was preliminarily verified by 2% agarose gel.

[0121] 4. Multiplex detection system PCR amplification: Mix the primers of CpG sites with good tissue specificity verified in two systems, and adjust the primer concentration to the best to achieve optimal amplification. The markers and primer concentrations in the two systems are shown in Table 5.

[0122] Table 5 Concentrations of 20 labeled primers

[0123]

[0124] The amount of each reagent added and the thermal cycle conditions in the multiplex PCR detection system are shown in Table 6 below:

[0125] Table 6 PCR multiplex amplification system

[0126]

[0127] The PCR thermal cycle conditions were as follows: 95°C, 10 min, 95°C, 20 s, 58°C, 1 min, 72°C, 30 s, for a total of 35 cycles; 72°C, 5 min.

[0128] Example 2: Detect CpG sites by single base extension and analyze methylation profiles.

[0129] The first step of purification: 2 μL of each of the multiplex amplification PCR products in the two systems in Example 1 were mixed, 1.25 μL SAP (shrimp alkaline phosphatase), 1 μL 10 × Cut Smart buffer, 0.3 μL Exo Ι (exonuclease Ι), 1 μL 10× EXO I buffer, and the reaction system was supplemented to 10 μL with water. Purification conditions: 37°C, 1h; 95°C, 15min.

[0130] CpG single base extension reaction: 5 μL of extension system, 2 μL of multiplex PCR purified product, 2 μL of SNaPshot Mix, 1 μL of extension primer, extension conditions: 96°C for 1 min; 96°C for 10 s, 50°C for 10 s, 60°C for 20 s, for a total of 40 cycles; 60°C for 1 min.

[0131] The second step of purification: add 1 μL SAP to 5 μL of extension product. Purification conditions: 37°C, 1h; 95°C, 15min.

[0132] Capillary electrophoresis and analysis.

[0133] Denaturation treatment system before electrophoresis: 1 μL purified product, 0.06 μL internal standard (GeneScan-LIZ 120), 10 μL formamide (Hi-Di formamide); treatment conditions: 95°C, 5 min, 0°C, 3 min.

[0134] The extension products were detected by ABI® 3500 DNA sequencer and analyzed by ABI ® GeneMapper IDV3.2.

[0135] Example 3: Detecting SNP molecular markers through single base extension and performing SNP personal typing.

[0136] Step 1: Purification: 2 μL of each of the multiplex amplification PCR products in the two systems in Example 1 were mixed, 1.25 μL SAP (shrimp alkaline phosphatase), 1 μL 10 × CutSmart buffer, 0.3 μL Exo Ι (exonuclease Ι), 1 μL 10 × EXO I buffer, and the reaction system was supplemented to 10 μL with water. Purification conditions: 37°C, 1h; 95°C, 15min.

[0137] CpG single base extension reaction: 5 μL of extension system, 2 μL of multiplex PCR purified product, 2 μL of SNaPshot Mix, 1 μL of extension primer, extension conditions: 96°C for 1 min; 96°C for 10 s, 50°C for 10 s, 60°C for 20 s, for a total of 40 cycles; 60°C for 1 min.

[0138] The second step of purification: add 1 μL SAP to 5 μL of extension product. Purification conditions: 37°C, 1h; 95°C, 15min.

[0139] Capillary electrophoresis and analysis.

[0140] Denaturation treatment system before electrophoresis: 1 μL purified product, 0.06 μL internal standard (GeneScan-LIZ 120), 10 μL formamide (Hi-Di formamide); treatment conditions: 95°C, 5 min, 0°C, 3 min.

[0141] The extension products were detected by ABI® 3500 DNA sequencer and analyzed by ABI ® GeneMapper IDV3.2.

[0142] Example 4: Population frequency survey.

[0143] 1. Collect individual specimens.

[0144] In this experiment, 114 samples of unrelated healthy individuals were collected, all of whom belonged to the Han population in Shanxi Province. Genomic DNA of blood samples was extracted using the Qiagen (DNeasy Blood & Tissue Kit) kit, and genomic DNA was quantified using the Invitrogen Qubit4. Afterwards, 300 ng of genomic DNA was subjected to bisulfite conversion using the Qiagen (EpiTect® Fast DNA BisμLfite Kit) kit.

[0145] Multiplex PCR amplification and detection

[0146] Using the converted DNA of 114 blood samples as templates, all samples were subjected to composite PCR amplification according to the multiplex detection system provided in Example 1, and the SNP individual typing of 113 samples was completed according to the SNP single base extension system provided in Example 3, and their allele frequencies were counted. Detailed information is shown in Table 7.

[0147] Table 7 Allele frequencies of 23 SNP markers in Shanxi population

[0148]

[0149] The final cumulative individual identification probability of the multiple detection system is 0.999983274, which is relatively high and can be used for individual identification of blood donors in mixed blood spots.

[0150] Example 5: Detection of old samples.

[0151] 1. Prepare DNA samples.

[0152] The genomic DNA of blood spot samples stored at room temperature for 2, 5, 7, and 9 months was extracted using the Qiagen DNeasy Blood & Tissue Kit, and the sample DNA was quantified using the Invitrogen Qubit4. Afterwards, 300 ng of genomic DNA was subjected to bisulfite conversion using the Qiagen EpiTect® Fast DNA BisμLfite Kit.

[0153] 2. Using the converted single-stranded DNA as a template, multiple PCR amplification was performed according to the method in Example 1, and CpG map analysis and SNP typing were performed according to the methods in Examples 2 and 3 to determine the stability of the system. Figure 3 ,4 The results showed that the methylation status of the blood spot samples stored at room temperature for 9 months remained unchanged, and the SNP typing was good, and the complete typing of the blood donors could be detected. The results showed that the kit of the present invention has good stability and can accurately and effectively identify the source of body fluids and perform individual identification after a long period of time.

[0154] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A kit for blood source identification and personal identification based on CpG-SNP linkage markers, characterized in that: The method comprises 23 pairs of specific multiplex PCR primers as shown in SEQ ID NO. 1 to 46, which are used to detect the following 23 markers, namely, 19 blood-specific CpG-SNP linked markers, 1 semen-specific CpG site, 1 saliva-specific CpG site, 1 vaginal secretion-specific CpG site, and 1 internal control, i.e., housekeeping gene, for evaluating the efficiency of bisulfite conversion. ACTB ; Among them, 19 blood-specific CpG-SNP linkage markers were cg19182633-rs2072110, cg01022219-rs4796991, cg04011671-rs4640436-rs72876044, cg06442294-rs10419232, cg11239720-rs58781231, cg01607849-rs12302749, cg19987129-rs6925308, cg09234599-rs11609960-rs10846771-rs7977743, and cg2412443 -rs68094061, cg03818715-rs2744388, cg15033269-rs587712, cg06645778-rs55988613, cg13581015-rs10848543, cg00578614-rs2523994, c g03666441-rs66687178, cg23927970-rs11827672, cg12237948-rs6132289, cg19092837-rs35111552-rs11121529, cg08792630-rs58775446; One semen-specific CpG site is cg13885748; One saliva-specific CpG site is cg26107890; One vaginal secretion-specific CpG site was cg09765089.

2. A kit for blood source identification and personal identification based on CpG-SNP linkage markers according to claim 1, characterized in that: The specific PCR primers for cg19182633-rs2072110 are the upstream primer of SEQ ID NO.1 and the downstream primer of SEQ ID NO.2; The specific PCR primers for cg01022219-rs4796991 are the upstream primer of SEQ ID NO.3 and the downstream primer of SEQ ID NO.4; The specific PCR primers for cg04011671-rs4640436-rs72876044 are the upstream primer of SEQ ID NO.5 and the downstream primer of SEQ ID NO.6; The specific PCR primers for cg06442294-rs10419232 are the upstream primer of SEQ ID NO.7 and the downstream primer of SEQ ID NO.8; The specific PCR primers for cg11239720-rs58781231 are the upstream primer of SEQ ID NO.9 and the downstream primer of SEQ ID NO.10; The specific PCR primers for cg01607849-rs12302749 are the upstream primer of SEQ ID NO.11 and the downstream primer of SEQ ID NO.12; The specific PCR primers for cg19987129-rs6925308 are the upstream primer of SEQ ID NO.13 and the downstream primer of SEQ ID NO.14; The specific PCR primers for cg09234599-rs11609960-rs10846771-rs7977743 are the upstream primer of SEQ ID NO.15 and the downstream primer of SEQ ID NO.16; The specific PCR primers for cg24124443-rs68094061 are the upstream primer of SEQ ID NO.17 and the downstream primer of SEQ ID NO.18; The specific PCR primers for cg03818715-rs2744388 are the upstream primer of SEQ ID NO.19 and the downstream primer of SEQ ID NO.20; The specific PCR primers for cg15033269-rs587712 are the upstream primer of SEQ ID NO.21 and the downstream primer of SEQ ID NO.22; The specific PCR primers for cg06645778-rs55988613 are the upstream primer of SEQ ID NO.23 and the downstream primer of SEQ ID NO.24; The specific PCR primers for cg13581015-rs10848543 are the upstream primer of SEQ ID NO.25 and the downstream primer of SEQ ID NO.26; The specific PCR primers for cg00578614-rs2523994 are the upstream primer of SEQ ID NO.27 and the downstream primer of SEQ ID NO.28; The specific PCR primers for cg03666441-rs66687178 are the upstream primer of SEQ ID NO.29 and the downstream primer of SEQ ID NO.30; The specific PCR primers for cg23927970-rs11827672 are the upstream primer of SEQ ID NO.31 and the downstream primer of SEQ ID NO.32; The specific PCR primers for cg12237948-rs6132289 are the upstream primer of SEQ ID NO.33 and the downstream primer of SEQ ID NO.34; The specific PCR primers for cg19092837-rs35111552-rs11121529 are the upstream primer of SEQ ID NO.35 and the downstream primer of SEQ ID NO.36; The specific PCR primers for cg08792630-rs58775446 are the upstream primer of SEQ ID NO.37 and the downstream primer of SEQ ID NO.38; The specific PCR primers for cg13885748 are the upstream primer of SEQ ID NO.39 and the downstream primer of SEQ ID NO.40; The specific PCR primers for cg26107890 are the upstream primer of SEQ ID NO.41 and the downstream primer of SEQ ID NO.42; The specific PCR primers for cg09765089 are the upstream primer of SEQ ID NO. 43 and the downstream primer of SEQ ID NO 44; ACTB The specific PCR primers are the upstream primer of SEQ ID NO.45 and the downstream primer of SEQ ID NO.

46.

3. A kit for blood source identification and personal identification based on CpG-SNP linkage markers according to claim 2, characterized in that: Also included are 22 CpG single base extension primers as shown in SEQ ID NOs. 47 to 68, corresponding to 19 blood-specific CpG-SNP linkage markers, 1 semen-specific CpG site, 1 saliva-specific CpG site, and 1 vaginal secretion-specific CpG site; Wherein: the extension primer sequence corresponding to cg19182633 is shown in SEQ ID NO.47; The extension primer sequence corresponding to cg01022219 is shown in SEQ ID NO.48; The extension primer sequence corresponding to cg04011671 is shown in SEQ ID NO.49; The extension primer sequence corresponding to cg06442294 is shown in SEQ ID NO.50; The extension primer sequence corresponding to cg11239720 is shown in SEQ ID NO.51; The extension primer sequence corresponding to cg01607849 is shown in SEQ ID NO.52; The extension primer sequence corresponding to cg19987129 is shown in SEQ ID NO.53; The extension primer sequence corresponding to cg09234599 is shown in SEQ ID NO.54; The extension primer sequence corresponding to cg24124443 is shown in SEQ ID NO.55; The extension primer sequence corresponding to cg03818715 is shown in SEQ ID NO.56; The extension primer sequence corresponding to cg15033269 is shown in SEQ ID NO.57; The extension primer sequence corresponding to cg06645778 is shown in SEQ ID NO.58; The extension primer sequence corresponding to cg13581015 is shown in SEQ ID NO.59; The extension primer sequence corresponding to cg00578614 is shown in SEQ ID NO.60; The extension primer sequence corresponding to cg03666441 is shown in SEQ ID NO.61; The extension primer sequence corresponding to cg23927970 is shown in SEQ ID NO.62; The extension primer sequence corresponding to cg12237948 is shown in SEQ ID NO.63; The extension primer sequence corresponding to cg19092837 is shown in SEQ ID NO.64; The extension primer sequence corresponding to cg08792630 is shown in SEQ ID NO.65; The extension primer sequence corresponding to cg13885748 is shown in SEQ ID NO.66; The extension primer sequence corresponding to cg26107890 is shown in SEQ ID NO.67; The extension primer sequence corresponding to cg09765089 is shown in SEQ ID NO.

68.

4. A kit for blood source identification and personal identification based on CpG-SNP linkage markers according to claim 3, characterized in that: Also included are 24 SNP single base extension primers as shown in SEQ ID NOs. 69 to 92, corresponding to 23 SNP molecular markers and housekeeping genes linked to 19 blood-specific CpG sites. ACTB ; The extension primer sequence corresponding to rs2072110 is as SEQ ID NO.69; The extension primer sequence corresponding to rs4796991 is as SEQ ID NO.70; The extension primer sequence corresponding to rs4640436 is as SEQ ID NO.71; The extension primer sequence corresponding to rs72876044 is as SEQ ID NO.72; The extension primer sequence corresponding to rs10419232 is as SEQ ID NO.73; The extension primer sequence corresponding to rs58781231 is as SEQ ID NO.74; The extension primer sequence corresponding to rs12302749 is as SEQ ID NO.75; The extension primer sequence corresponding to rs6925308 is as SEQ ID NO.76; The extension primer sequence corresponding to rs11609960 is as SEQ ID NO.77; The extension primer sequence corresponding to rs10846771 is as SEQ ID NO.78; The extension primer sequence corresponding to rs7977743 is as SEQ ID NO.79; The extension primer sequence corresponding to rs68094061 is as SEQ ID NO.80; The extension primer sequence corresponding to rs2744388 is as SEQ ID NO.81; The extension primer sequence corresponding to rs587712 is as SEQ ID NO.82; The extension primer sequence corresponding to rs55988613 is as SEQ ID NO.83; The extension primer sequence corresponding to rs10848543 is as SEQ ID NO.84; The extension primer sequence corresponding to rs2523994 is as SEQ ID NO.85; The extension primer sequence corresponding to rs66687178 is as SEQ ID NO.86; The extension primer sequence corresponding to rs11827672 is as SEQ ID NO.87; The extension primer sequence corresponding to rs6132289 is as SEQ ID NO.88; The extension primer sequence corresponding to rs35111552 is as SEQ ID NO.89; The extension primer sequence corresponding to rs11121529 is as SEQ ID NO.90; The extension primer sequence corresponding to rs58775446 is as SEQ ID NO.91; ACTB The corresponding extension primer sequence is shown as SEQ ID NO.

92.

5. A kit for blood source identification and personal identification based on CpG-SNP linkage markers according to claim 4, characterized in that: Each pair of specific multiplex PCR primers contains at least one SNP linked to a blood-specific CpG site and at most three SNPs.

6. A kit for blood source identification and personal identification based on CpG-SNP linkage markers according to claim 4, characterized in that: The kit also includes deionized formamide and molecular weight internal standard GeneScan-120 LIZ for detecting single base extension products.

7. A kit for blood source identification and personal identification based on CpG-SNP linkage markers as claimed in any one of claims 1 to 6, characterized in that: The steps include: 1) extracting the DNA of the sample to be tested as a template, treating it with bisulfite, and using 23 pairs of specific multiplex PCR primers as shown in SEQ ID NOs. 1 to 46 to obtain amplification products by multiplex specific PCR; 2) Purifying the amplified product, and obtaining the extension product by a single base extension system using 22 CpG single base extension primers as shown in SEQ ID NOs. 47 to 68 and 24 SNP single base extension primers as shown in SEQ ID NOs. 69 to 92; 3) The extended products are purified and analyzed using capillary electrophoresis, 3500 sequencer and gene analysis software to complete blood source identification and personal identification.

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