An 8CpG panel test kit and two prediction models for body fluid classification

Abstract

The present invention belongs to the application field of molecular biotechnology and forensic evidence science, discloses an 8CpG panel detection kit and two prediction models for body fluid classification, and specifically discloses a DNA methylation marker for identifying the type of body fluid specimen. The body fluid-specific expression difference DNA methylation marker provided by the present invention is obtained by systematic screening of a group of healthy young and middle-aged Han people (20 to 45 years old) that is more common in forensic practice in my country. The DNA methylation marker can simultaneously accurately classify and identify five body fluids of the human body (venous blood, saliva, semen, menstrual blood, and vaginal secretions). At the same time, the above-mentioned body fluid-specific DNA methylation marker has good specificity and stability in a healthy young and middle-aged Han population, and has great application prospects in forensic practice in my country.

Description

Technical Field

[0001] The invention belongs to the application fields of molecular biotechnology and forensic evidence science, and specifically relates to an 8CpG panel detection kit and two prediction models for body fluid classification. Background Art

[0002] In forensic identification, body fluid samples are a common type of biological evidence. Clarifying the source type of body fluid tissue extracted from the crime scene or the victim is helpful to determine the nature of the case, infer the course of the crime and recreate the scene, especially in sexual assault cases. However, in addition to some body fluids with similar properties (such as venous blood and menstrual blood, etc.) that are difficult to distinguish with the naked eye, some trace or old body fluid stains often do not have typical body fluid properties and complete characteristic cell structures, and their tissue source type cannot be directly or indirectly determined by physical methods such as naked eye observation or microscopic observation. At the same time, even if the tissue source of body fluid samples can be directly inferred through naked eye observation and experience, it is necessary to identify them with scientific basis before they can be used as evidence in court trials. Therefore, the establishment of identification and analysis methods for the source of different types of body fluid tissue types has always been a scientific problem that forensic evidence researchers have been committed to solving.

[0003] DNA methylation can change the functional state of gene regulatory regions without changing the genomic nucleotide sequence. During development, different tissues and cells in the same organism undergo different remethylation processes, and ultimately express different DNA methylation patterns. This pattern is relatively stable and heritable, thus forming tissue-specific differentially methylated regions. Studies have revealed that it is one of the ideal molecular markers for body fluid traceability. There are various methods for detecting DNA methylation, among which the methylation-sensitive single nucleotide primer extension (Ms-SnuPE) technology is a site-specific DNA methylation analysis technology based on a capillary electrophoresis platform. It not only achieves docking with the forensic grassroots laboratory platform, but also realizes simultaneous quantitative detection of multiple DNA methylation sites, greatly improving the detection efficiency, which is conducive to promoting the application and transformation of DNA methylation research results in forensic practice.

[0004] In 2014, Park et al. screened out 8 DNA methylation sites with high sensitivity and body fluid specificity based on the whole genome methylation chip Illumina Human Methylation 450KBeadchip. In 2015, Lee et al. screened out 64 potential body fluid-specific differentially expressed DNA methylation sites from more than 450,000 methylation sites in the whole genome based on the same chip technology. These studies have confirmed that by expanding the screening range of body fluid-specific differentially expressed methylation sites, new and more stable potential body fluid-specific differentially expressed DNA methylation sites can be selected, and a multiple Ms-SnuPE system with application potential can be constructed. However, although many studies have proved that DNA methylation sites have stable and heritable characteristics, as a new potential molecular marker for body fluid identification, it still needs to be systematically evaluated for application to analyze its stability as a molecular marker for forensic identification. The above-mentioned studies and technologies are currently mostly carried out on foreign populations, and the age information of the samples is unknown. my country is a vast country, and the Han nationality is a populous ethnic group in my country. Therefore, the stability of some reported DNA methylation sites in different groups in my country may be insufficient, and their application potential in forensic practice in my country is still unclear. In particular, the difference in DNA methylation expression between the two vaginal body fluids (i.e., vaginal secretions and menstrual blood) is small. Current research has not yet found ideal and stable DNA methylation sites specific to these two body fluids. Some scholars even combined the two into one vaginal body fluid for research. In addition, after the construction of the multiple Ms-SnuPE system, how to scientifically interpret its test data to accurately determine the body fluid tissue source of the sample is another problem faced by forensic practice. Therefore, the determination of body fluid identification results based on DNA methylation test data still needs in-depth research. Summary of the invention

[0005] The purpose of the first aspect of the present invention is to provide a DNA methylation marker for identifying the type of body fluid sample.

[0006] The purpose of the second aspect of the present invention is to provide the use of the DNA methylation marker of the first aspect of the present invention in the identification of body fluid samples.

[0007] The purpose of the third aspect of the present invention is to provide a reagent for detecting the DNA methylation marker of the first aspect of the present invention.

[0008] The fourth aspect of the present invention aims to provide a DNA methylation marker detection kit.

[0009] The purpose of the fifth aspect of the present invention is to provide the use of the reagent of the third aspect of the present invention and / or the kit of the fourth aspect of the present invention in the identification of the type of body fluid sample.

[0010] The sixth aspect of the present invention aims to provide a method for identifying the tissue origin of a body fluid sample.

[0011] The object of the seventh aspect of the present invention is to provide a prediction model for determining the results of the identification method of the sixth aspect of the present invention.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] In a first aspect of the present invention, a DNA methylation marker for identifying the type of body fluid sample is provided, wherein the DNA methylation marker is selected from at least one of the methylation sites indicated by CG in the sequences of SEQ ID NO.1 to SEQ ID NO.8, or selected from at least one of the corresponding methylation sites in the fully complementary sequences of SEQ ID NO.1 to SEQ ID NO.8.

[0014] Preferably, the DNA methylation marker includes at least one of cg24301930, cg25922751, cg03902386, cg03282313, cg05614346, cg24772753, cg05558714 and cg07452397.

[0015] Preferably, the body fluid is a human body fluid.

[0016] Preferably, the body fluid is one or more of human venous blood, human saliva, human semen, human menstrual blood and human vaginal secretions.

[0017] The second aspect of the present invention provides use of the DNA methylation marker of the first aspect of the present invention in identification of body fluid samples.

[0018] Preferably, the body fluid is a human body fluid.

[0019] Preferably, the body fluid is one or more of human venous blood, human saliva, human semen, human menstrual blood and human vaginal secretions.

[0020] The third aspect of the present invention provides a reagent for detecting the DNA methylation marker of the first aspect of the present invention.

[0021] Preferably, the reagents include an amplification primer and a single-base extension primer.

[0022] Preferably, the amplification primers and single-base extension primers include:

[0023] The nucleotide sequences shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:25 for cg24301930;

[0024] The nucleotide sequences shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:26 for cg25922751;

[0025] The nucleotide sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 27 for cg03902386;

[0026] The nucleotide sequences shown in SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:28 for cg03282313;

[0027] The nucleotide sequences shown in SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:29 for cg05614346;

[0028] The nucleotide sequences shown in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:30 for cg24772753;

[0029] The nucleotide sequences shown in SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:31 for cg05558714;

[0030] The nucleotide sequences shown in SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:32 for cg07452397.

[0031] The fourth aspect of the present invention provides a kit comprising the reagent of the third aspect of the present invention.

[0032] Preferably, the kit further comprises at least one of Master mix, rSAP, Exonuclease I and SNaPshot Multiplex Mix.

[0033] Preferably, the kit further comprises an internal quality control C.

[0034] Further preferably, the amplification primers for the internal quality control C are the nucleotide sequences shown in SEQ ID NO:33 to SEQ ID NO:34.

[0035] Further preferably, the single base extension primer of the internal quality control C is the nucleotide sequence shown in SEQ ID NO:35.

[0036] The fifth aspect of the present invention provides use of the reagent of the third aspect of the present invention and / or the kit of the fourth aspect of the present invention in identification of the type of body fluid specimens.

[0037] Preferably, the body fluid is a human body fluid.

[0038] Preferably, the body fluid is one or more of human venous blood, human saliva, human semen, human menstrual blood and human vaginal secretions.

[0039] A sixth aspect of the present invention provides a method for identifying the tissue origin of a body fluid sample, comprising the steps of using the reagent of the third aspect of the present invention and / or the kit of the fourth aspect of the present invention.

[0040] Preferably, the identification method specifically comprises the following steps:

[0041] (1) Extract DNA from body fluid samples and convert DNA methylation to C / T;

[0042] (2) using the amplification primers of the third aspect of the present invention to amplify the DNA site converted by methylation C / T;

[0043] (3) using the single-base extension primer of the third aspect of the present invention to perform single-base extension on the amplification product of step (2), and analyzing it by capillary electrophoresis;

[0044] (4) Determine the type of body fluid sample based on the capillary electrophoresis analysis results.

[0045] Preferably, step (1) uses a Chelex-100 method or a magnetic bead method to extract DNA from body fluid samples.

[0046] Preferably, the methylated C / T conversion in step (1) is performed by converting the DNA using bisulfite.

[0047] Preferably, the amplification conditions in step (2) are 90-96°C for 1-3 minutes; 90-95°C for 25-35 seconds, 50-60°C for 80-100 seconds, 70-73°C for 25-35 seconds, 30-35 cycles; 55-65°C for 25-30 minutes. Further, 90-95°C for 2-3 minutes; 90-95°C for 30-35 seconds, 50-56°C for 80-90 seconds, 70-72°C for 30-35 seconds, 30-35 cycles; 60-65°C for 25-30 minutes. Further, 95°C for 2 minutes; 95°C for 30 seconds, 56°C for 90 seconds, 72°C for 30 seconds, 35 cycles; 60°C for 30 minutes.

[0048] Preferably, the amplification reaction system in step (2) comprises Master mix, PCR Primer mix, and methylated C / T converted DNA; the PCR Primer mix is ​​a mixture of the amplification primers of the third aspect of the present invention, and the final concentration of each primer in the reaction system is 0.077 μmol / L.

[0049] Preferably, the amplified product in step (2) is purified before the product is subjected to a single base extension reaction.

[0050] More preferably, the purification reaction system comprises rSAP and Exonuclease I; further comprises 0.2-0.3U rSAP and 0.1-0.3U Exonuclease I.

[0051] More preferably, the purification conditions are 30-37°C for 1-1.5 hours and 75-82°C for 20 minutes; further preferably, 37°C for 1 hour and 80°C for 20 minutes.

[0052] Preferably, the extension reaction conditions described in step (3) are 90-97°C for 5-15 seconds, 45-55°C for 5-10 seconds, 57-65°C for 25-35 seconds, and 20-30 cycles; further, 96-97°C for 10-15 seconds, 45-50°C for 5-10 seconds, 60-65°C for 30-35 seconds, and 20-25 cycles; further, 96°C for 10 seconds, 50°C for 5 seconds, 60°C for 30 seconds, and 25 cycles.

[0053] Preferably, the reaction system for the amplification in step (3) comprises SNaPshot Multiplex Mix and SBEPrimer Mix, and the SBE Primer Mix is ​​a mixture of the single base extension primers of the third aspect of the present invention, wherein the final concentration of the single base extension primers of the DNA methylation markers cg25922751, cg24301930, cg03282313, cg24772753 and cg05614346 in the reaction system is 0.066 μmol / L, the final concentration of the single base extension primers of the DNA methylation marker cg03902386 in the reaction system is 0.099 μmol / L, and the final concentration of the single base extension primers of the DNA methylation markers cg05558714 and cg07452397 in the reaction system is 0.033 μmol / L.

[0054] Preferably, the product obtained by the single base extension amplification is purified before the capillary electrophoresis analysis.

[0055] Further preferably, the purified reaction system comprises rSAP.

[0056] More preferably, the reaction conditions for the purification are 30-37°C for 1-1.5 hours and 75-82°C for 20 minutes; further preferably, 37°C for 1 hour and 80°C for 20 minutes.

[0057] Preferably, the reaction system of the capillary electrophoresis analysis comprises a product obtained by single base extension amplification, formamide and GeneScan 120LIZ dye molecular weight standard.

[0058] Preferably, the capillary electrophoresis analysis system is placed on ice for 2 to 5 minutes after being heated at 90 to 95° C. for 3 to 8 minutes.

[0059] Preferably, the conditions for the capillary electrophoresis analysis are: a hot plate temperature of 60° C., an injection voltage of 2 KV, an injection time of 12 seconds, an electrophoresis voltage of 15 KV, and an electrophoresis time of 25 minutes.

[0060] Preferably, the result determination is performed using an artificial classification prediction model and / or a random forest classification prediction model.

[0061] The seventh aspect of the present invention provides a prediction model for determining the results of the identification method of the sixth aspect of the present invention, wherein the prediction model is an artificial classification prediction model and / or a random forest classification prediction model.

[0062] Preferably, the body fluid identification pattern diagram of the artificial classification prediction model is as follows Figure 5 shown.

[0063] Preferably, the application of the random forest classification prediction model relies on the randomForest package of the R language, the random forest model type is classification, the number of trees is 100, and the split point of the variable is 2. Part of the code for the application of the random forest classification prediction model is as follows Figure 7 As shown in the figure, the data set included in the model construction is as follows Figure 8 shown.

[0064] The beneficial effects of the present invention are:

[0065] The body fluid-specific differentially expressed DNA methylation marker provided by the present invention is obtained by systematically screening a group of healthy young and middle-aged Han people (20 to 45 years old) that is more common in forensic practice in my country; the DNA methylation marker can simultaneously accurately classify and identify five body fluids (venous blood, saliva, semen, menstrual blood, and vaginal secretions). At the same time, the body fluid-specific DNA methylation marker has good specificity and stability in the healthy young and middle-aged Han Chinese population, and has great application prospects in forensic practice in my country.

[0066] The amplicon length of the body fluid-specific differentially expressed DNA methylation marker provided by the present invention is controlled within 200 bp, and can be applied to typing and identification of degraded biological samples.

[0067] Through testing and evaluation, the identification method of body fluid samples provided by the present invention successfully detected 0.5ng of DNA after conversion, and there was no significant difference in DNA methylation rate, which has high sensitivity. At the same time, the system is not interfered by common animal DNA such as dogs, cats, rats, mice and pigeons, and has good species specificity.

[0068] The present invention constructs an artificial body fluid prediction model and a random forest machine learning body fluid prediction model that can efficiently analyze the detection data of the system. The output results of the two models verify each other to achieve artificial intelligence precise tracing of five body fluids (venous blood, semen, saliva, menstrual blood and vaginal secretions) involving healthy young and middle-aged Han people in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 This is the capillary electrophoresis diagram of the single base extension product of the complex system.

[0070] Figure 2 The DNA methylation levels of DNA of different concentrations were determined using the identification method of Example 3; wherein A is the DNA methylation level of venous blood, B is the DNA methylation level of saliva, C is the DNA methylation level of menstrual blood, D is the DNA methylation level of semen, and E is the DNA methylation level of vaginal secretions.

[0071] Figure 3 This is the specificity evaluation result of the identification method of Example 3.

[0072] Figure 4 The results of electrophoresis are for typing differentially expressed DNA methylation sites in body fluids.

[0073] Figure 5 The figure shows the body fluid identification pattern of the artificially set classification threshold and classification prediction model.

[0074] Figure 6 The results of DNA methylation levels of mixed samples of DNA from different body fluids using the identification method of Example 7; wherein A is the result of DNA methylation levels of mixed samples of semen and menstrual blood, B is the result of DNA methylation levels of semen and saliva, and C is the result of DNA methylation levels of semen and vaginal secretions.

[0075] Figure 7 Part of the code for the random forest classification prediction model.

[0076] Figure 8This is the RF-100.csv file used in the code for the random forest classification prediction model. DETAILED DESCRIPTION

[0077] The present invention is further described in detail below through specific examples.

[0078] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0079] The materials and reagents used in this example, unless otherwise specified, were obtained from commercial sources. The Master Mix (Platinum Multiplex PCR Master Mix) was purchased from Applied Biosystems. TM , Catalog No. 4464268; Shrimp Alkaline Phosphatase (rSAP) was purchased from New England Biolabs. The product number is M0371S; Exonuclease I was purchased from Takara, the product number is 2650A; SNaPshot MultiplexKit was purchased from Applied Biosystems TM , Cat. No. 4323159; GeneScan TM 120LIZ TM Dye molecular weight standards were purchased from Applied Biosystems TM , item number 4324287.

[0080] In order to achieve artificial intelligence precise tracing of five types of body fluids (venous blood, semen, saliva, menstrual blood and vaginal secretions) involving healthy young and middle-aged Han people in my country, the present invention systematically selected body fluid-specific differentially expressed DNA methylation sites within the human genome, designed a pair of PCR amplification primers and a single base extension primer for each DNA methylation site, and used a multiple Ms-SnuPE system to obtain the C / T value (forward extension) or G / A value (reverse extension), which is the ratio of methylation to non-methylation, after electrophoresis separation and fluorescence intensity measurement of the terminal extension products on a capillary electrophoresis platform. The methylation rate of each DNA methylation site in the test fragment was analyzed, and an internal quality control C (single base on a specific fragment) was introduced into the system to control the efficiency of DNA bisulfite conversion. At the same time, the inventors constructed an artificial body fluid classification prediction model and a random forest body fluid classification prediction model respectively. The coordinated application and joint research of the two are expected to achieve accurate tissue tracing of five types of body fluids (venous blood, semen, saliva, menstrual blood and vaginal secretions).

[0081] Example 1 Systematic selection and verification of differentially expressed DNA methylation sites in body fluids

[0082] The inventors used the Illumina Infinium Methylation EPIC Beadchip technology to detect the methylation expression levels of more than 850,000 methylation sites in the whole genome of five types of body fluids (venous blood, semen, saliva, menstrual blood, and vaginal secretions). The methylation expression levels in the target body fluids and other non-target body fluids were significantly different, and the absolute value of the difference was greater than 0.2. The standard was used to systematically select body fluid-specific differentially expressed DNA methylation sites, and verified by pyrophosphate sequencing, and finally 8 body fluid-specific differentially expressed DNA methylation sites were obtained. The specific information of the 8 body fluid-specific differentially expressed DNA methylation sites is shown in Table 1.

[0083] Table 1 Detailed information of 8 differentially expressed DNA methylation sites in body fluids

[0084]

[0085] Example 2 Design of amplification primers and single base extension primers for each DNA methylation site

[0086] The key points to consider when designing primers include: 1) primer length; 2) primer GC content; 3) primer Tm value; 4) whether the primer binding region contains genetic polymorphic variant sites (SNP / DIP); 5) the sequence of the primer itself, whether it will form a hairpin structure, etc. In addition, considering the applicability of the composite amplification system to old and degraded samples that may be encountered in forensic practice, the size of the amplicon needs to be controlled within 200bp. At the same time, a single base C that is completely unmethylated on a specific fragment is selected and introduced into the detection system as an internal quality control for testing the efficiency of DNA bisulfite conversion, so as to control the efficiency of DNA bisulfite conversion. The information of primers designed for 8 CpG sites is shown in Table 2.

[0087] Table 2 Primer information of 8 body fluid-specific differentially expressed DNA methylation sites

[0088]

[0089]

[0090] Example 3 Construction of a multiplex Ms-SnuPE (8CpG panel) system based on specific differentially expressed DNA methylation sites and identification method of unknown body fluids

[0091] On the basis of Example 2, a single base C (IQ) on a specific fragment was used as an internal quality control base (upstream primer: 5'-GTYGTTAGGTAGTAGTATTAGTAGGTTTAGT-3' (SEQ ID NO: 33), downstream primer: 5'-CAATACAAATAATATATCAAAAAACCA-3' (SEQ ID NO: 34), single base extension primer: 5'-(CT) 12 TAGGTAGTAGTATTAGTAGGTTTAG-3'(SEQ ID NO:35)), construct a multiple Ms-SnuPE system. The primers of a single body fluid-specific differentially expressed DNA methylation site were diluted, and primers of equal molar mass were mixed for each site to prepare a primer mixture. The primer mixture includes PCR Primer Mix and SBE Primer Mix, PCR Primer Mix is ​​a specific amplification primer for body fluid-specific differentially expressed DNA methylation sites, and SBE Primer Mix is ​​a specific single base extension primer for 8 body fluid-specific differentially expressed DNA methylation sites. The final concentration of each primer is shown in Table 3.

[0092] Table 3 Primer concentrations for amplification of 8 differentially expressed DNA methylation sites in body fluids

[0093]

[0094] The method for identifying unknown body fluids based on a multiplex Ms-SnuPE (8CpG panel) system of specifically expressed differential DNA methylation sites includes the following steps:

[0095] (1) DNA extraction

[0096] The DNA was analyzed using the QIAamp DNA Investigator Kit (Qiagen, Cat. No. 56504) or PrepFiler TM BTAForensic DNA Extraction Kit (Applied Biosystems TM , catalog number 4463352) to extract genomic DNA from body fluids (such as venous blood, semen, saliva, vaginal secretions and menstrual blood).

[0097] (2) DNA methylation C / T conversion

[0098] The DNA in (1) was subjected to bisulfite conversion using EpiTect Fast DNA Bisulfite Kit (Qiagen, Catalog No. 59824) or EZ DNA Methylation Kit (Zymo, Catalog No. D5001) to obtain methylated C / T converted DNA.

[0099] (3) PCR amplification

[0100] The DNA converted to methylated C / T was used as the DNA template for PCR amplification, and the primers in Table 1 were used to perform multiplex PCR amplification on eight loci including cg24301930, cg25922751, cg03902386, cg03282313, cg05558714, cg07452397, cg05614346 and cg24772753 and internal control C. The reaction system for multiplex PCR amplification was as follows: Master Mix 10 μL, PCR Primer Mix 2 μL, DNA converted to bisulfite 1-6 μL, Nuclease-Free Water to 20 μL, and the amplification reaction conditions were as follows: 95°C for 2 minutes; 95°C for 30 seconds, 56°C for 90 seconds, 72°C for 30 seconds, 35 cycles; 60°C for 30 minutes.

[0101] (4) Purification of PCR amplification products

[0102] The PCR amplification product obtained in (3) was purified, and the purification system was as follows: 5.2 μL of PCR amplification product, 2.5 μL of rSAP (1 U / μL), 1 μL of 10×rSAP buffer, 0.3 μL of Exonuclease I (5 U / μL), and 1 μL of 10×Exonuclease I buffer. The prepared reaction system was incubated at 37°C for 1 hour, incubated at 80°C for 20 minutes to inactivate the enzyme, stored at 4°C, and the next step was performed within 1 hour.

[0103] (5) Single base extension

[0104] The purified PCR amplification product in (4) was subjected to a multiplex system single base extension to obtain a multiplex system single base extension product. The reaction system for the multiplex system single base extension was: 5 μL of SNaPshot Multiplex Mix, 1 μL of SBE PrimerMix, 1 to 4 μL of purified PCR amplification product, and 10 μL of Nuclease-Free Water. The reaction conditions for the multiplex system single base extension were: 96°C for 10 s, 50°C for 5 s, 60°C for 30 s, and 25 cycles.

[0105] (6) Purification of single-base extension products

[0106] 1 μL of rSAP and 1.2 μL of 10×rSAP buffer were added to the single base extension product of the composite system for purification reaction. The purification reaction conditions were: 37°C for 1 hour, 80°C for 20 minutes, and stored at 4°C for later use.

[0107] (7) Capillary electrophoresis analysis

[0108] Take 2 μL of the purified complex system single base extension product, mix it with 2 μL of formamide and 0.5 μL of GeneScan120LIZ dye molecular weight standard, denature at 95°C for 5 minutes, place it on ice for 3 minutes, put it in a capillary electrophoresis instrument (including but not limited to 3100 series, 3130 series, 3500 series genetic analyzers), and perform capillary electrophoresis under the following conditions: injection voltage is 2KV, injection time is 12 seconds; electrophoresis voltage is 15KV, and electrophoresis time is 25 minutes. After the electrophoresis is completed, use ID-X software processes the detected data and obtains the typing map and typing data of the DNA methylation site, and calculates the methylation rate of the DNA methylation site according to formula (1) or (2). Formula (1): methylation rate = C peak height / (C peak height + T peak height) × 100%, formula (2): methylation rate = G peak height / (G peak height + A peak height) × 100%.

[0109] Example 4

[0110] By using the identification method of Example 3, five types of human body fluids (venous blood, semen, saliva, vaginal secretions and menstrual blood, all of which are body fluids of healthy young and middle-aged Han people (20 to 45 years old) in my country, provided by volunteers who have passed ethical approval) were tested. The results are as follows: Figure 1 As shown in the figure, the single base extension of the 8 humor-specific differentially expressed DNA methylation sites can detect effective peak types; the above 8 humor-specific differentially expressed DNA methylation sites are divided into two groups: forward extension (C: yellow / black peak; T: red peak) and reverse extension (G: blue peak; A: green peak). The typing results of the five human body fluids are shown in Figure 4 As shown, it is shown that the identification method of Example 3 can accurately identify five human body fluids, including human venous blood, semen, saliva, vaginal secretions and menstrual blood.

[0111] Example 5 Sensitivity Evaluation

[0112] The identification method of Example 3 was used to detect human body fluids (venous blood, saliva, semen, vaginal secretions or menstrual blood), and 0.5ng, 1ng, 2ng, 5ng and 10ng of methylated C / T converted DNA were used as templates for detection to evaluate the sensitivity of the detection method of Example 3.

[0113] The results are as follows Figure 2 As shown, after methylated C / T conversion, DNA above 0.5 ng was successfully detected, and there was no significant difference in the DNA methylation rate of each concentration, which confirmed that the identification method has high sensitivity.

[0114] Example 6 Specificity Assessment

[0115] The identification method of Example 3 is used to detect DNA in human body fluids (venous blood, saliva, semen, vaginal secretions or menstrual blood) and DNA in common animals such as dogs, cats, rats, mice and pigeons.

[0116] The results are as follows Figure 3 As shown, only human body fluid DNA showed effective peaks, while other animal DNA did not show effective peaks, indicating that the identification method of Example 3 has strong specificity.

[0117] Example 7

[0118] The unknown body fluid is tested using the identification method of Example 3, and the test result is determined using one of the following two determination methods, thereby identifying the unknown body fluid.

[0119] (1) Manual classification prediction model

[0120] Based on the pyrophosphate sequencing results of 100 body fluid samples, the inventors set up a body fluid identification pattern diagram of the artificial classification prediction model, such as Figure 5 For each body fluid, if one or more of the two specific sites meets the classification threshold condition, a judgment can be made.

[0121] (2) Random Forest Classification Prediction Model

[0122] The application of this model relies on the randomForest package of R language. The random forest model type is classification, the number of trees is 100, and the split point of the variable is 2. Some codes for the application of the random forest classification prediction model are as follows Figure 7 As shown, the data set included in the model construction (RF-100.csv file) ( Figure 8), the inventors previously used pyrosequencing to detect the methylation expression levels of the 8 body fluid-specific differentially expressed DNA methylation sites in 100 different body fluid samples, extracted 90% of the data as a training set to construct a random forest classification prediction model that can be used to identify the five body fluid types, and used the remaining 10% of the data as a validation set to test the accuracy of the model.

[0123] Example 8

[0124] 95 body fluid samples (body fluid samples of healthy young and middle-aged Han Chinese (20-45 years old) in my country, provided by volunteers who passed ethical approval) were tested using the detection method of Example 3 and the identification method of Example 7, and the test results were judged based on 1) the manual classification prediction model and 2) the random forest classification prediction model, respectively.

[0125] According to 1) the classification threshold of each body fluid-specific CpG site, the test results were judged by the manual classification prediction model. It was finally found that only 4 vaginal fluid samples were not successfully classified among the 95 samples, and the overall classification accuracy of the system was 95.8%. According to 2) the random forest classification prediction model, the test results were judged and predicted. All 95 body fluid samples were successfully predicted, indicating that the random forest prediction accuracy was 100% and the Kappa value was 1.

[0126] The methylated C / T converted DNA of some samples was further mixed with semen and menstrual blood, semen and saliva, and semen and vaginal fluid at a DNA mass ratio of 1:1, 1:2, and 1:4 (three replicates per group) to test the identification efficiency of the identification method of Example 7 (determination by 1) artificial classification prediction model) for mixed body fluid samples. The results showed that all 27 samples could be successfully identified as mixed samples ( Figure 6 ).

[0127] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of a reagent or kit in identification of body fluid specimen types; The reagent or kit includes an amplification primer and a single-base extension primer for detecting DNA methylation sites; The DNA methylation sites are cg24301930, cg25922751, cg03902386, cg03282313, cg05614346, cg24772753, cg05558714, and cg07452397; The body fluid is one or more of human venous blood, human saliva, human semen, human menstrual blood and human vaginal secretions; The amplification primers and single base extension primers are: The nucleotide sequences shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:25 for cg24301930; The nucleotide sequences shown in SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:26 for cg25922751; The nucleotide sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 27 for cg03902386; The nucleotide sequences shown in SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:28 for cg03282313; The nucleotide sequences shown in SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:29 for cg05614346; The nucleotide sequences shown in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:30 for cg24772753; The nucleotide sequences shown in SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:31 for cg05558714; and The nucleotide sequences shown in SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:32 for cg07452397.

2. The use according to claim 1, characterized in that: The kit further comprises at least one of Master mix, rSAP, Exonuclease I and SNaPshot Multiplex Mix.

3. A method for identifying the source of a body fluid sample tissue, comprising the step of detecting the body fluid sample tissue using the reagent or kit described in claim 1 or 2; the body fluid is one or more of human venous blood, human saliva, human semen, human menstrual blood and human vaginal secretions.

4. The identification method according to claim 3, characterized in that: The identification method specifically comprises the following steps: (1) Extract DNA from body fluid samples and perform DNA methylation C / T conversion; (2) using the amplification primers described in claim 1 or 2 to amplify the site of the DNA converted by methylation C / T; (3) using the single base extension primer described in claim 1 or 2 to perform single base extension on the amplification product of step (2), and analyzing it by capillary electrophoresis; (4) Determine the type of body fluid sample based on the capillary electrophoresis analysis results.

5. The identification method according to claim 4, characterized in that: The amplification conditions in step (2) are 90-96°C for 1-3 minutes; 90-95°C for 25-35 seconds, 50-60°C for 80-100 seconds, 70-73°C for 25-35 seconds, 30-35 cycles; 55-65°C for 25-30 minutes.

6. The identification method according to claim 4, characterized in that: The single base extension reaction conditions described in step (3) are 90-97°C for 5-15 seconds, 45-55°C for 5-10 seconds, 57-65°C for 25-35 seconds, and 20-30 cycles.

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