A primer set for individual identification of hair shaft, a kit and application thereof

Abstract

The application belongs to the technical field of forensic medicine, and particularly relates to a primer group for hair shaft individual identification, a kit and application thereof. The primer group can realize detection of cSNP and / or MH sites on a high-expression mRNA marker of a hair shaft, solve the problem that a hair shaft sample cannot be subjected to individual identification, and has high precision. Meanwhile, the instrument and reagent used in the application are commonly equipped in a forensic laboratory, and the operation is simple and easy to popularize.

Description

Technical Field

[0001] This invention belongs to the field of forensic medicine technology, specifically relating to a primer set, reagent kit, and their applications for individual hair shaft identification. Background Technology

[0002] Hair samples are one of the most common samples in forensic identification work. Forensic workers often collect hair samples at crime scenes. However, since most hair falls out naturally, the hair follicles are often atrophied or absent, which can easily lead to test failure or insufficient nuclear DNA.

[0003] Since hair shafts also contain a certain amount of DNA, although the amount is extremely small and much of it is degraded, some methods for individual identification based on hair shafts still exist in current research, such as autosomal short tandem repeat (STR) detection, mitochondrial DNA detection, and single amino acid polymorphism (SNP) detection. However, due to the trace degradation of hair shaft DNA, autosomal STR detection often results in long-segment locus loss or allele insertion. Although detecting two hypervariable regions in mitochondrial DNA can reveal differences in single nucleotide polymorphisms (SNPs), since mitochondrial DNA is maternally inherited, it can only be used to exclude individuals from different maternal lines. While SNP methods can infer non-synonymous SNPs from genomic DNA sequences based on changes in SNPs, synonymous mutations in DNA are not reflected in single amino acids, resulting in low information content. Furthermore, chemical or biological modifications of amino acids can cause mass shifts during mass spectrometry analysis, leading to errors in inferring non-synonymous SNPs.

[0004] It is evident that existing methods for individual identification based on hair shafts all have some insurmountable defects, and there is a lack of new genetic markers and detection methods in this field to achieve accurate individual identification of hair shaft samples. Summary of the Invention

[0005] The purpose of this invention is to provide a primer set, a kit, and their applications for individual hair shaft identification. The primer set and the kit containing the primer set can detect cSNP and / or MH sites on highly expressed mRNA markers in hair shafts, solving the problem of individual identification of hair shaft samples with high accuracy.

[0006] This invention provides reagents for detecting human gene cSNP sites in one or more of the following applications: mRNA polymorphism detection for individual hair shaft 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 table below:

[0007]

[0008]

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

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

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

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

[0013] RNA was extracted from the hair shaft sample to be tested, and the obtained hair shaft sample RNA was reverse transcribed to obtain hair shaft sample cDNA.

[0014] The hair shaft sample cDNA was amplified by multiplex PCR using the primer set described in the above technical solution to obtain the multiplex PCR amplification product of the hair shaft sample.

[0015] Library construction was performed based on the multiplex PCR amplification products of the hair shaft sample to obtain the hair shaft sample sequencing library.

[0016] The hair shaft sample sequencing library was subjected to high-throughput sequencing to obtain high-throughput sequencing results;

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

[0018] Beneficial effects:

[0019] This invention provides reagents for detecting human gene cSNP sites in one or more of the following applications: mRNA polymorphism detection for individual hair shaft identification, forensic identification of hair shaft samples, and forensic testing of hair shaft samples; the human gene cSNP sites include 404 cSNPs. Based on this, this invention also provides primer sets and kits capable of amplifying the 404 cSNPs, as well as applications using RNA as a template in mRNA polymorphism detection for individual hair shaft identification, forensic identification of hair shaft samples, and forensic testing of hair shaft samples.

[0020] Compared to existing methods that use hair shaft DNA as a template for individual detection, this invention uses RNA as a template. Even if the hair shaft is a slightly degraded sample, the RNA is multi-copy, and the hair shaft contains sufficient RNA molecules, providing a sufficient template for subsequent detection and overcoming the problem of not being able to obtain sufficient nuclear DNA from the hair shaft. Furthermore, the RNA molecules contain polymorphic sites that can be used for individual identification, namely the 404 cSNP sites. The RNA sequence retains the polymorphism of the DNA sequence, thus RNA polymorphism can be used to reflect DNA polymorphism. Moreover, the primer set described in this invention can achieve the detection of cSNPs and / or MH sites on highly expressed mRNA markers in the hair shaft, solving the problem of not being able to identify individuals from hair shaft samples, and with high accuracy. At the same time, the instruments and reagents used in this invention are all standard equipment in forensic laboratories, are simple to operate, and are easy to promote. Detailed Implementation

[0021] This invention provides reagents for detecting human gene cSNP sites in one or more of the following applications: mRNA polymorphism detection for individual hair shaft identification, forensic identification of hair shaft samples, and forensic testing of hair shaft samples; the human gene cSNP sites include 404 cSNPs. A table containing the 404 cSNPs has been presented in the invention description section and will not be repeated here.

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

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

[0024] This invention also provides a kit for individual hair shaft identification, the kit comprising the primer set described in the above-described technical solution. In one embodiment, the kit may further comprise RNA extraction-related reagents and / or RNA reverse transcription-related reagents; in another embodiment, the kit may further comprise RNA extraction-related reagents and RNA reverse transcription-related reagents; in yet another embodiment, the kit may further comprise high-throughput library preparation and / or sequencing-related reagents; and in yet another embodiment, the kit may further comprise high-throughput library preparation and sequencing-related reagents. This invention does not impose any special limitations on the source and specifications of the RNA extraction-related reagents, RNA reverse transcription-related reagents, and high-throughput library preparation and sequencing-related reagents; commercially available reagents in the art can be used.

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

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

[0027] RNA was extracted from the hair shaft sample to be tested, and the obtained hair shaft sample RNA was reverse transcribed to obtain hair shaft sample cDNA.

[0028] The hair shaft sample cDNA was amplified by multiplex PCR using the primer set described in the above technical solution to obtain the multiplex PCR amplification product of the hair shaft sample.

[0029] Library construction was performed based on the multiplex PCR amplification products of the hair shaft sample to obtain the hair shaft sample sequencing library.

[0030] The hair shaft sample sequencing library was subjected to high-throughput sequencing to obtain high-throughput sequencing results;

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

[0032] As one implementation method, the genotyping method of the present invention can refer to the data analysis workflow 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 IntGenet. 2024 Jul; 71:103066. doi:10.1016 / j.fsigen.2024.103066.Epub 2024May31.PMID:38833776.]. 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 implementation method, the amplicon read count threshold 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] This invention does not impose any special limitations on the processes of RNA extraction, RNA transcription, multiplex PCR amplification, and library construction; these processes and methods are familiar to those skilled in the art. This invention also does not impose any special requirements on the high-throughput sequencing platform; any conventional high-throughput sequencing platform in the art can be selected based on the adapter used for library construction. For example, in this specific embodiment, the BGI DNBSEQ platform is used.

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

[0035] Example 1

[0036] An mRNA polymorphism detection site and its primer sequence for individual identification of hair shaft samples are provided, consisting of the following sites and primers: the sites are shown in Table 1 (the rs number of 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 shows the information on the selected genes and cSNP sites.

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[0047] Table 2 Primer sequences for the multiplex amplification system

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

[0058] Example 2

[0059] A high-throughput detection method for RNA polymorphism in individual hair shaft samples, comprising the following steps:

[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 part about 1 cm from the hair follicle end. Collect about 20 mg in total and place it in a 1.5 mL enzyme-free centrifuge tube. Use scissors to cut the hair shaft into fragments of about 3 mm. Wash with anhydrous ethanol, enzyme-free water and anhydrous ethanol in sequence, and let stand and air dry.

[0062] (2) Grinding: Grind the hair shaft obtained in step (1) using a cryogenic grinder with the following settings: 60Hz, 60s, 2 times.

[0063] (3) Digestion: The hair shaft after grinding in step (2) was digested with 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). The constant temperature metal bath was set as follows: 12000 rpm, 58℃ for 5-10 min.

[0064] (4) RNA extraction: Total RNA was extracted from the sample obtained in step (3) using RNAiso Plus reagent. The specific procedure 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 min;

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

[0067] 3) Centrifuge at 4℃ and 12000 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 inverting, and precipitate at -20°C for 2 hours;

[0069] 5) Centrifuge at 4℃, 12000rpm for 20min, discard the supernatant, add 1mL of 75% ethanol, invert several times, centrifuge at 4℃, 12000rpm for 20min, and discard the supernatant.

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

[0071] (5) RNA reverse transcription: The HiFiScript gDNARemoval RTMasterMix reverse transcription kit was used.

[0072] 2. Design of High-Throughput Detection System

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

[0074] The high-throughput multiplex detection system was designed by Shanghai Diwin Biotechnology Co., Ltd., and consists of a total of 326 amplicones, 61 of which contain two or more cSNP sites. This invention refers to them as microhaplotypes (MH). Primer sequences are shown in Table 2.

[0075] 3. Construction of high-throughput sequencing libraries

[0076] Use the QuarMultiple PCR Library Construction Kit manual to complete library construction.

[0077] 4 sequencing

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

[0079] 5 Results Analysis

[0080] Since the hair shaft was a trace amount of degraded material, the amplicon read count threshold was set to 30 during the results analysis. Following the data analysis workflow in the reference [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 numbers in Table 1) as reference sequences to obtain the cSNP / MH site genotyping.

[0081] Example 3

[0082] 1. High-throughput detection kit for hair shaft RNA polymorphism in Shanxi population

[0083] Forty hair shaft samples were collected from Shanxi Province. RNA was extracted, reverse transcribed, library constructed, sequenced, and analyzed according to the method described in Example 2 to obtain population data for each locus in this composite detection kit. Analysis revealed that 97 amplicones in this kit did not meet the read count >30 standard in all samples. Ultimately, 286 cSNP loci on 71 genes were identified, contained in 229 amplicones, including 45 MH loci. Among the loci meeting the analysis criteria, 18 amplicones showed no polymorphism in the population, while the remaining amplicones showed 2-5 alleles. Allele frequencies and genotype frequencies are shown in Tables 3-4.

[0084] Table 3. Allele frequencies at cSNP / MH loci

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

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[0100] Due to significant differences among the various hair shaft samples, the highest number of amplicon detected in the 40 samples tested was 218, the lowest was 84, with an average of 167 ± 27. The random matching probability of the system was 4.71 × 10⁻⁶. -30 ~2.67×10 -110 This meets the requirements for individual identification.

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

[0102] Three samples were collected: hair shafts (head hair, armpit hair, leg hair, and pubic hair), totaling 12 samples. The genotypes of the armpit hair, leg hair, and pubic hair shafts were compared with those of the head hair shaft using the detection system established in Example 2. The results are shown in Table 5. It was found that head hair and pubic hair shafts yielded more genotypes than armpit hair and leg hair shafts, but the genotypes of the detected genotypes were consistent.

[0103] Table 5. Results of hair shaft testing at different locations

[0104]

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

[0106] Two samples, one black and one white, were collected and tested using the detection system established in Example 2. The results are shown in Table 6. It was found that more than 141 amplicones could be detected in the black hair shaft and more than 161 amplicones could be detected in the white hair shaft, with a total of 135-153 amplicones detected. All the amplicon genotypes were consistent.

[0107] Table 6. Detection results of hair shafts of different colors

[0108] sample Black feathers White hair The cSNP / MH was detected in total cSNP / MH with the same subtype Sample 1 160 199 153 153 Sample 2 141 161 135 135

[0109] 4. Species-specific detection

[0110] Hair shaft samples were collected from four common animals: rabbit, mouse, cat, and dog. The detection system established in Example 2 was used for analysis. The results are shown in Table 7. The extracted RNA mass was 2.34 ± 0.38 μg, indicating that the RNA extraction method for human hair shaft samples is also applicable to animal hair shaft samples. Some amplicons were also detected in animal hair shaft samples, with the highest number (82) detected in dog hair shaft samples. However, the number of reads for these amplicons was significantly lower than in human hair shaft samples, and the cSNP / MH genotype differed from the reference sequence.

[0111] Table 7 Results of Hair Root Detection in Species

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

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

[0114] Hair shafts from three samples were collected and stored at room temperature for 6 months, 1 year, and 2 years, respectively, and detected using the detection system described in Example 2. The results are shown in Table 8. It was found that there was little difference in the detection of hair shafts stored for 6 months compared to fresh hair shafts, but the number of amplicon detected decreased in hair shafts stored for 1-2 years.

[0115] Table 8. Detection results of old hair dryer samples

[0116]

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

[0118] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of reagents for detecting human gene cSNP sites in the detection of mRNA polymorphisms for individual hair shaft identification or in the forensic identification of hair shaft samples; the human gene cSNP sites include the 404 cSNPs shown in the table below: ； The reagents include a set of primers for amplifying the 404 cSNP sites; The primer set comprises 326 primer pairs, the nucleotide sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.

652.

2. The application according to claim 1, characterized in that, The reagents also include RNA extraction reagents and / or RNA reverse transcription reagents.

3. A method for detecting mRNA polymorphisms for individual hair shaft identification, characterized in that, Includes the following steps: RNA was extracted from the hair shaft sample to be tested, and the obtained hair shaft sample RNA was reverse transcribed to obtain hair shaft sample cDNA. Multiplex PCR amplification of the hair shaft sample cDNA was performed using the primer set to obtain the multiplex PCR amplification product of the hair shaft sample; the primer set includes 326 primer pairs, and the nucleotide sequences of the 326 primer pairs are shown in SEQ ID NO.1~SEQ ID NO.652; Library construction was performed based on the multiplex PCR amplification products of the hair shaft sample to obtain the hair shaft sample sequencing library. The hair shaft sample sequencing library was subjected to high-throughput sequencing to obtain high-throughput sequencing results; Genotyping was performed based on the high-throughput sequencing results to obtain cSNP and / or microhaplotype typing results.

4. The method for detecting mRNA polymorphism according to claim 3, characterized in that, The threshold for the number of amplicon reads during genotyping was set to 30.

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