Primer group and kit for individual identification of hair shaft and application of primer group and kit

Abstract

The invention belongs to the technical field of forensic medicine, and particularly relates to a primer group and a kit for individual recognition of hair shafts and application of the primer group and the kit. The primer group provided by the invention can realize detection of cSNP and / or MH sites on a hair stem high-expression mRNA marker, solves the problem that a hair stem sample cannot be subjected to individual recognition, and is relatively high in accuracy. Meanwhile, instruments and reagents used in the method are conventionally prepared in a forensic laboratory, operation is easy and convenient, and popularization is easy.

Description

Technical Field

[0001] The invention belongs to the technical field of forensic medicine, and in particular relates to a primer set and a kit for hair shaft individual identification and applications thereof. Background Art

[0002] Hair samples are one of the common samples in forensic identification work. Forensic workers often collect hair samples at the crime scene, but because most hair is shed naturally, the hair follicles are often atrophied or absent, which can easily lead to detection failure or insufficient nuclear DNA.

[0003] Since hair shafts also contain a certain amount of DNA, although the DNA content in hair shafts is extremely small and mostly degraded, there are some methods for individual identification based on hair shafts in existing research, such as autosomal short tandem repeat (STR) detection, mitochondrial DNA detection, and single amino acid polymorphism detection. However, due to the current situation of slight degradation of hair shaft DNA, the autosomal STR detection method often shows the phenomenon of long-segment locus loss, segment locus allele loss or insertion; although the difference of single nucleotide polymorphism can be obtained by detecting the two highly variable regions of mitochondrial DNA, since mitochondrial DNA is maternally inherited, it can only be used to exclude individuals from different maternal lines; although the single amino acid polymorphism method can infer the non-synonymous single nucleotide polymorphism of genomic DNA sequence through the change of single amino acid polymorphism, the synonymous mutation on DNA cannot be reflected on the single amino acid, the information content is low, and the chemical modification or biological modification of amino acids causes mass shift during mass spectrometry analysis, which will cause errors in the inference of non-synonymous single nucleotide polymorphisms.

[0004] It can be seen that the existing methods of individual identification based on hair shafts have some insurmountable defects, and the field still lacks new genetic markers and detection methods for achieving accurate individual identification of hair shaft samples. Summary of the invention

[0005] The purpose of the present invention is to provide a primer set, a kit and its application for hair shaft individual identification. The primer set and the kit comprising the primer set can realize the detection of cSNP and / or MH sites on hair shaft highly expressed mRNA markers, solve the problem that hair shaft samples cannot be individually identified, and have high accuracy.

[0006] The present invention provides a reagent for detecting human gene cSNP sites for use in one or more of mRNA polymorphism detection for hair shaft individual identification, forensic identification of hair shaft samples, and forensic testing of hair shaft samples; the human gene cSNP sites include 404 cSNPs as shown in the following table:

[0007]

[0008]

[0009] The present invention also provides a primer set for hair shaft individual identification, the primer set comprising 326 primer pairs, the nucleotide sequences of the 326 primer pairs are shown in SEQ ID NO.1 to SEQ ID NO.652.

[0010] The present invention also provides a kit for identifying individual hair shafts, the kit comprising the primer set described in the above technical solution.

[0011] The present invention also provides the use of the primer set described in the above technical solution or the kit described in the above technical solution in one or more of the mRNA polymorphism detection for hair shaft individual identification, forensic identification of hair shaft samples and forensic detection of hair shaft samples.

[0012] The present invention also provides a method for detecting mRNA polymorphism for detecting hair shaft individual identification, comprising the following steps:

[0013] Extracting RNA from the hair shaft sample to be tested, and reverse transcribing the obtained hair shaft sample RNA to obtain hair shaft sample cDNA;

[0014] Performing multiple PCR amplification on the hair shaft sample cDNA using the primer set described in the above technical solution to obtain multiple PCR amplification products of the hair shaft sample;

[0015] constructing a library based on the multiplex PCR amplification products of the hair shaft samples to obtain a hair shaft sample sequencing library;

[0016] Performing high-throughput sequencing on the hair shaft sample sequencing library to obtain high-throughput sequencing results;

[0017] Genotyping is performed based on the high-throughput sequencing results to obtain cSNP and / or microhaplotype typing results.

[0018] Beneficial effects:

[0019] The present invention provides the use of a reagent for detecting human gene cSNP sites in one or more of mRNA polymorphism detection for hair shaft individual identification, forensic identification of hair shaft samples, and forensic detection of hair shaft samples; the human gene cSNP sites include 404 cSNPs. On this basis, the present invention also provides a primer set and a kit capable of amplifying the 404 cSNPs, and the use of RNA as a template in mRNA polymorphism detection for hair shaft individual identification, forensic identification of hair shaft samples, and forensic detection of hair shaft samples.

[0020] Compared with the method of using hair shaft DNA as a template for individual detection in the prior art, the present invention uses RNA as a template for detection. In this way, even if the hair shaft is a trace amount of degraded sample, the RNA is multi-copy, and there are sufficient RNA molecules in the hair shaft to provide sufficient templates for subsequent detection, overcoming the problem that sufficient nuclear DNA cannot be obtained from the hair shaft; in addition, there are polymorphic sites that can be used for individual identification on the RNA molecule, namely the 404 cSNP sites, and the polymorphism of the DNA sequence is retained on the RNA sequence, so the RNA polymorphism can be used to react with the DNA polymorphism. Furthermore, the primer set of the present invention can realize the detection of cSNP and / or MH sites on the hair shaft highly expressed mRNA marker, solving the problem that the hair shaft sample cannot be used for individual identification, and the accuracy is high. At the same time, the instruments and reagents used in the present invention are all routinely equipped in forensic laboratories, which are simple to operate and easy to promote. DETAILED DESCRIPTION

[0021] The present invention provides a reagent for detecting human gene cSNP sites for use in one or more of mRNA polymorphism detection for hair shaft individual identification, forensic identification of hair shaft samples, and forensic detection of hair shaft samples; the human gene cSNP sites include 404 cSNPs. A table containing the 404 cSNPs has been presented in the invention content section and will not be repeated here.

[0022] As one embodiment, the reagent of the present invention includes a primer set for amplifying the 404 cSNP sites; as another embodiment, the primer set includes 326 pairs of primers, and the nucleotide sequences of the 326 pairs of primers are shown in SEQ ID NO.1 to SEQ ID NO.652.

[0023] The present invention also provides a primer set for hair shaft individual identification, the primer set comprising 326 primer pairs, the nucleotide sequences of the 326 primer pairs are shown in SEQ ID NO.1 to SEQ ID NO.652.

[0024] The present invention also provides a kit for hair shaft individual identification, the kit comprising the primer set described in the above technical solution. As an embodiment, the kit of the present invention may also include RNA extraction related reagents and / or RNA reverse transcription related reagents; as another embodiment, the kit may also include RNA extraction related reagents and RNA reverse transcription related reagents; as another embodiment, the kit of the present invention may also include high-throughput library construction and / or sequencing related reagents; as another embodiment, the kit of the present invention may also include high-throughput library construction and sequencing related reagents. The present invention does not specifically limit the sources and specifications of the RNA extraction related reagents, RNA reverse transcription related reagents, and high-throughput library construction and sequencing related reagents, and reagents from commercial sources in the field may be used.

[0025] The present invention also provides the use of the primer set described in the above technical solution or the kit described in the above technical solution in one or more of the mRNA polymorphism detection for hair shaft individual identification, forensic identification of hair shaft samples and forensic detection of hair shaft samples.

[0026] Specifically, the present invention also provides a method for detecting mRNA polymorphism for detecting hair shaft individual identification, comprising the following steps:

[0027] Extracting RNA from the hair shaft sample to be tested, and reverse transcribing the obtained hair shaft sample RNA to obtain hair shaft sample cDNA;

[0028] Performing multiple PCR amplification on the hair shaft sample cDNA using the primer set described in the above technical solution to obtain multiple PCR amplification products of the hair shaft sample;

[0029] constructing a library based on the multiplex PCR amplification products of the hair shaft samples to obtain a hair shaft sample sequencing library;

[0030] Performing high-throughput sequencing on the hair shaft sample sequencing library to obtain high-throughput sequencing results;

[0031] Genotyping is performed based on the high-throughput sequencing results to obtain cSNP and / or microhaplotype typing results.

[0032] As an embodiment, the genotyping method of the present invention can refer to the data analysis process in the literature [Liu Z, Wang J, Li L, Yang H, Yu H, Fan J, Zhang M, Zhang Y, Liu J, Li Z, Zhang G. Identification of the body fluid donor in mixtures through target mRNA cSNP sequencing. Forensic Sci Int Genet. 2024 Jul; 71: 103066. doi: 10.1016 / j.fsigen.2024.103066. Epub 2024 May 31. PMID: 38833776.], and the reference sequence used is the transcript data of the gene where the 404 cSNPs are located, and the relevant information of the gene and its transcript is recorded in Table 1. As another embodiment, the threshold of the number of amplicon reads in the genotyping process of the present invention is set to 30, instead of the conventional threshold of 200-500, to match the nature of the hair shaft sample as a micro-degraded sample.

[0033] The present invention has no special restrictions on the process of RNA extraction, RNA transcription, multiplex PCR amplification, and library construction, and can be operated according to the process and method well known to those skilled in the art. The present invention has no special requirements for the high-throughput sequencing platform, and a conventional high-throughput sequencing platform in the art can be selected according to the adapter used for library construction. For example, in a specific embodiment, the BGI DNBSEQ platform is used.

[0034] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail with reference to the following embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Example 1

[0036] An mRNA polymorphism detection site and primer sequence for individual identification of hair shaft samples, comprising the following sites and primers: the sites are shown in Table 1 (the rs number of the cSNP in Table 1 is the accession number in the Ensembl database and the NCBI database), and the primers are shown in Table 2.

[0037] Table 1 Screened genes and cSNP loci information

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[0040]

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[0047] Table 2 Primer sequences of composite amplification system

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[0053]

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[0057] In Table 2, the upstream primer sequence and the downstream primer sequence in the second row are numbered as SEQ ID NO.1 and SEQ ID NO.2, respectively, and the upstream primer sequence and the downstream primer sequence in the third row are numbered as SEQ ID NO.3 and SEQ ID NO.4, respectively. The upstream primer sequence and the downstream primer sequence in the last row are numbered as SEQ ID NO.651 and SEQ ID NO.652, respectively.

[0058] Example 2

[0059] A high-throughput detection method for RNA polymorphism for individual identification of hair shaft samples, the steps are as follows:

[0060] 1. Extraction and reverse transcription of hair shaft RNA

[0061] (1) Pretreatment: Cut a hair shaft sample of about 5 cm in length and cut off the 1 cm portion near the hair follicle end, collecting a total of about 20 mg and placing it in a 1.5 mL enzyme-free centrifuge tube. Use scissors to cut the hair shaft into fragments of about 3 mm, wash them with anhydrous ethanol, enzyme-free water, and anhydrous ethanol in turn, and let them stand to dry.

[0062] (2) Grinding: Grind the hair shaft obtained in step (1) using a cryo-grinder, with the setting conditions being: 60 Hz, 60 s, 2 times.

[0063] (3) Digestion: Use 230 μl of digestion solution (50 μl 1% SDS, 50 μl 1M Tris-HCl, 50 μl 0.5M EDTA, 50 μl 3% NaCl, 20 μl 1M DTT, 10 μl pk) to digest the hair shaft ground in step (2). The constant temperature metal bath setting conditions are: rotation speed 12000 rpm, 58°C 5-10 min.

[0064] (4) RNA extraction: Use RNAiso Plus reagent to extract the total RNA of the sample obtained in step (3). The specific operation is as follows:

[0065] 1) Add 1 mL of RNAiso Plus reagent to the digested enzyme-free centrifuge tube, mix manually, and let stand for 5 minutes;

[0066] 2) Add 200 μl of chloroform, mix thoroughly, and let stand for 10 min;

[0067] 3) Centrifuge at 4°C, 12,000 rpm for 15 min. After centrifugation, aspirate the supernatant and transfer it to a new enzyme-free centrifuge tube;

[0068] 4) Add an equal volume of isopropanol, mix by inversion, and precipitate at -20°C for 2 hours;

[0069] 5) Centrifuge at 4°C, 12,000 rpm for 20 min, discard the supernatant, add 1 mL of 75% ethanol, invert several times, centrifuge at 4°C, 12,000 rpm for 20 min, and discard the supernatant;

[0070] 6) Open the lid of the enzyme-free centrifuge tube, dry it at room temperature, and add 20 μl of enzyme-free water to dissolve the RNA.

[0071] (5) RNA reverse transcription: Use HiFiScript gDNARemoval RTMasterMix reverse transcription kit.

[0072] 2. Design of high-throughput detection system

[0073] In this example, 78 hair shaft highly expressed mRNAs were screened from the existing whole transcriptome sequencing data, including 404 coding SNP (cSNP) sites. The site information is shown in Table 1.

[0074] The high-throughput composite detection system was designed by Shanghai Diying Biotechnology Co., Ltd., with a total of 326 amplicons, 61 of which contained 2 or more cSNP sites, which are referred to as microhaplotypes (MH) in the present invention. The primer sequences are shown in Table 2.

[0075] 3 High-throughput sequencing library construction

[0076] The library construction was completed using the QuarMultiple PCR library construction kit operating manual.

[0077] 4 Sequencing

[0078] Sequencing was performed using the DNBSEQ platform and PE150 mode.

[0079] 5 Results Analysis

[0080] Since hair shafts are trace-degraded samples, the threshold for amplicon reads was set to 30 during result analysis. According to the data analysis process in the literature [Liu Z, Wang J, Li L, Yang H, Yu H, Fan J, Zhang M, Zhang Y, Liu J, Li Z, Zhang G. Identification of the body fluid donor in mixtures through target mRNA cSNP sequencing. Forensic Sci Int Genet. 2024 Jul; 71: 103066. doi: 10.1016 / j.fsigen.2024.103066. Epub 2024 May 31. PMID: 38833776.], the sequencing results were analyzed using the transcriptome sequences of all genes (the sequences corresponding to NM in Table 1) as reference sequences to obtain the typing of cSNP / MH sites.

[0081] Example 3

[0082] 1. Polymorphism detection of hair shaft RNA high-throughput detection kit in Shanxi population

[0083] 40 hair shaft samples were collected in Shanxi Province, and RNA was extracted, reverse transcribed, library constructed, sequenced and analyzed according to the method in Example 2 to obtain the population data of each site in this composite detection kit. After analysis, in this kit, 97 amplicons did not meet the standard of reads number > 30 in all samples. Finally, 286 cSNP sites on 71 genes remained, which were included in 229 amplicons, of which 45 were MH. Among the sites that met the analysis criteria, 18 amplicons were not observed to be polymorphic in the population, and 2-5 alleles were observed in the remaining amplicons. The allele frequencies and genotype frequencies are shown in Tables 3 and 4.

[0084] Table 3 Allele frequencies of cSNP / MH loci

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[0091] Table 4 Genotype frequencies of RNA cSNP / MH markers

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[0100] Due to the large differences between different hair shaft samples, among the 40 samples tested this time, the maximum number of amplicons detected was 218, the minimum was 84, and the average was 167±27. The random matching probability of the system was 4.71×10 -30 ~2.67×10 -110 , meeting individual identification requirements.

[0101] 2.Consistency test of hair shaft typing results in different parts of the body

[0102] The hair shafts of the head, armpit hair shafts, leg hair shafts, and pubic hair shafts of 3 samples were collected, for a total of 12 samples, and the typing of the armpit hair shafts, leg hair shafts, and pubic hair shafts was compared with the typing of the hair shafts. The results are shown in Table 5, and it was found that the hair shafts and pubic hair shafts could obtain more marker typing than the armpit hair shafts and leg hair shafts, but the typing of the markers detected in total was consistent.

[0103] Table 5 Test results of hair shafts at different locations

[0104]

[0105] 3. Detection of hair shaft typing results of different colors

[0106] Black hair shafts and white hair shafts of 2 samples were collected and tested using the detection system established in Example 2. The results are shown in Table 6, and it was found that more than 141 amplicons could be detected in black hair shafts, more than 161 amplicons could be detected in white hair shafts, and a total of 135-153 amplicons were detected, and the typing of all the amplicons detected was consistent.

[0107] Table 6 Test results of hair shafts of different colors

[0108] sample Black hair shaft White hair shaft Co-detected cSNP / MH cSNP / MH with the same genotype Sample 1 160 199 153 153 Sample 2 141 161 135 135

[0109] 4. Species-specific detection

[0110] Hair shaft samples of four common animals were collected, including rabbit, mouse, cat, and dog. The detection system established in Example 2 was used for detection. The results are shown in Table 7, and it was found that the mass of the extracted RNA was 2.34±0.38μg. The RNA extraction method of human hair shaft samples is also applicable to animal hair shaft samples. Some amplicons can also be detected in animal hair shaft samples. The most amplicons were detected in dog hair shaft, which was 82, but the number of reads of the amplicons was much lower than that of human hair shaft samples, and the typing of cSNP / MH was different from the reference sequence.

[0111] Table 7 Species hair shaft test results

[0112] Species cSNP rabbit 49 mouse 44 cat 51 dog 82

[0113] 5. Test results of old hair shaft samples

[0114] Hair shafts from 3 samples were collected and placed at room temperature for 6 months, 1 year and 2 years respectively, and tested using the detection system in Example 2. The results are shown in Table 8, and it was found that there was little difference between the detection of hair shafts placed for 6 months and fresh hair shafts, but the number of amplicons detected in hair shafts placed for 1-2 years decreased.

[0115] Table 8 Test results of old hair shaft samples

[0116]

[0117] It can be concluded from the above embodiments that the detection kit of the present invention has great application potential, and can be used for detection in hair shaft samples in different states, and the site typing detected in hair shafts of different parts, different colors or different storage times is consistent. This also proves that the detection kit of the present invention has high accuracy in individual identification using hair shaft samples as detection samples.

[0118] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting human gene cSNP sites in one or more of mRNA polymorphism detection for hair shaft individual identification, forensic identification of hair shaft samples, and forensic testing of hair shaft samples; the human gene cSNP sites include 404 cSNPs as shown in the following table:

2. The use according to claim 1, characterized in that: The reagents include a primer set for amplifying the 404 cSNP sites.

3. The use according to claim 2, characterized in that: The primer set includes 326 pairs of primers, and the nucleotide sequences of the 326 pairs of primers are shown as SEQ ID NO.1 to SEQ ID NO.

652.

4. A primer set for hair shaft individual identification, characterized in that: The primer set includes 326 pairs of primers, and the nucleotide sequences of the 326 pairs of primers are shown as SEQ ID NO.1 to SEQ ID NO.

652.

5. A kit for identifying individual hair shafts, characterized in that: The kit comprises the primer set according to claim 4.

6. The kit according to claim 5, characterized in that The kit also includes RNA extraction related reagents and / or RNA reverse transcription related reagents.

7. The kit according to claim 6, characterized in that The kit also includes high-throughput library construction and / or sequencing related reagents.

8. Use of the primer set of claim 4 or the kit of any one of claims 5 to 7 in one or more of the following: detection of mRNA polymorphism for hair shaft individual identification, forensic identification of hair shaft samples, and forensic testing of hair shaft samples.

9. A method for detecting mRNA polymorphism for detecting individual identification of hair shafts, characterized in that: The steps include: Extracting RNA from the hair shaft sample to be tested, and reverse transcribing the obtained hair shaft sample RNA to obtain hair shaft sample cDNA; Performing multiplex PCR amplification on the hair shaft sample cDNA using the primer set of claim 4 to obtain multiplex PCR amplification products of the hair shaft sample; constructing a library based on the multiplex PCR amplification products of the hair shaft samples to obtain a hair shaft sample sequencing library; Performing high-throughput sequencing on the hair shaft sample sequencing library to obtain high-throughput sequencing results; Genotyping is performed based on the high-throughput sequencing results to obtain cSNP and / or microhaplotype typing results.

10. The mRNA polymorphism detection method according to claim 9, characterized in that: The threshold of the number of amplicon reads during genotyping was set to 30.

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