A sperm-specific mRNA-cSNP primer composition, kit and method based on SNaPshot technology

Abstract

This invention discloses a semen-specific mRNA-cSNP primer composition, kit, and method based on SNaPshot technology, belonging to the field of molecular identification technology. The primer composition provided by this invention includes amplification primers with nucleotide sequences as shown in SEQ ID NO. 1-34 and single-base extension primers as shown in SEQ ID NO. 35-52. This invention further provides a method for identifying human semen samples, including extracting sample mRNA, reverse transcription to obtain cDNA, performing an amplification reaction using amplification primers, and then performing a single-base extension reaction using single-base extension primers. Experimental results show that the identification method of this invention has advantages such as high specificity, high sensitivity, and good reproducibility, and can accurately identify individual semen donors, which is of great significance for achieving accurate semen-related criminal investigations and forensic identification.

Description

Technical Field

[0001] This invention relates to the field of molecular identification technology, and in particular to a sperm-specific mRNA-cSNP primer composition, kit, and method based on SNaPshot technology. Background Technology

[0002] In forensic biological evidence practice, the identification and examination of semen or semen stains is a crucial step in the analysis of physical evidence in sexual assault cases, and is essential for determining the nature of the case and clarifying the direction of the investigation. Especially in criminal sexual assault cases, the detection of semen stains is directly related to the determination of biological evidence of the occurrence of sexual acts. However, male semen often mixes with female vaginal secretions and other bodily fluids to form mixed stains, greatly increasing the difficulty of separating and identifying semen components. Therefore, establishing accurate and sensitive methods for semen (stain) examination has always been a core research topic in this field.

[0003] Traditional semen (stain) identification techniques mainly rely on morphological observation, serological tests, biochemical analysis, and immunological detection. While these techniques offer advantages such as ease of operation and rapid detection, their limitations are also significant: First, the specificity of traditional semen stain identification methods is generally low due to interference from factors such as disease state, cross-reactivity between body fluids, and the subjectivity of result interpretation; second, they require large quantities of sample, making it difficult to meet the testing needs of trace or degraded semen stain samples. In recent years, significant advances in molecular biology have opened up new technical pathways for semen (stain) identification. Studies have shown that differential methylation analysis, microbiome analysis, copy number variation (CNV) analysis, and RNA analysis all demonstrate significant advantages in forensic semen (stain) identification. Among these, the detection technology based on messenger RNA (mRNA) has developed particularly rapidly. Semen (stain) identification based on semen-specific mRNA markers has been proven to have high specificity and accuracy by using capillary electrophoresis (CE) or next-generation sequencing (NGS) platforms. The corresponding result analysis algorithms are also becoming increasingly mature, providing strong support for forensic applications.

[0004] In body fluid (spot) identification, the conventional procedure requires extracting mRNA and DNA from the same sample for separate testing. Tissue-specific expression profiling based on mRNA is used to determine the body fluid type, while STR typing of DNA is used for individual identification. However, special body fluids such as semen often appear in mixed sample (spot) identification scenarios. STR typing results may originate from body fluid or tissue components that mRNA analysis failed to identify, leading to erroneous association between DNA and RNA evidence. To explore identification strategies more suitable for single and mixed body fluids (spots), scientists have proposed utilizing coding region single nucleotide polymorphisms (cSNPs) in body fluid-specific mRNAs to directly link the detected body fluid type to the genetic polymorphism information of its donor. Existing research has strongly demonstrated the significant application potential of cSNP markers in body fluid (spot) identification, especially providing a new approach to overcome the challenge of identifying different body fluid donor sources in mixed samples. This aims to simultaneously determine the body fluid type and obtain the SNP genetic information of the individual from that body fluid source.

[0005] Currently, SNP detection mainly relies on technologies such as gene chips, NGS, and SNaPshot. While gene chips offer high throughput, they suffer from limitations including complex procedures, difficult result interpretation, and high chip fabrication costs. Similarly, NGS technology has stringent requirements for equipment and involves complex data analysis processes. In contrast, SNaPshot technology significantly reduces detection costs due to its compatibility with existing CE platforms; secondly, its data analysis process is relatively simplified, facilitating routine laboratory operations; and most importantly, compared to gene chip technology, SNaPshot technology avoids non-specific reactions that may occur during probe hybridization, thus effectively reducing false positive and false negative results. Summary of the Invention

[0006] The purpose of this invention is to provide a semen-specific mRNA-cSNP primer composition, kit, and method based on SNaPshot technology to solve the problems existing in the prior art. The identification method provided by this invention has the advantages of high specificity, high sensitivity, and good reproducibility, and can accurately identify individual semen donors, which is of great significance for achieving accurate semen-related criminal investigations and forensic identification.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides a primer composition for detecting semen-specific mRNA-cSNP markers, comprising amplification primers and single-base extension primers;

[0009] The amplification primers include primers with nucleotide sequences as shown in SEQ ID NO.1-34;

[0010] The single-base extension primers include primers with nucleotide sequences as shown in SEQ ID NO.35-52.

[0011] The present invention also provides the application of the above-described primer composition in the preparation of products for detecting semen-specific mRNA-cSNP markers.

[0012] Furthermore, the product is a reagent kit.

[0013] The present invention also provides a kit for detecting semen-specific mRNA-cSNP markers, comprising the primer composition described above.

[0014] Furthermore, it also includes amplification reaction reagents and single-base extension reaction reagents.

[0015] The present invention also provides the application of the above-described primer composition or the above-described kit in the preparation of products for identifying semen.

[0016] Furthermore, the identification of semen includes determining the semen tissue type and obtaining the SNP genotyping of the semen donor.

[0017] The present invention also provides the application of the above-described primer composition or the above-described kit in the identification of semen.

[0018] Furthermore, the identification of semen includes determining the semen tissue type and obtaining the SNP genotyping of the semen donor.

[0019] This invention also provides a method for obtaining SNP genotyping of sperm donors, comprising the following steps:

[0020] mRNA was extracted from the sample to be tested, reverse transcribed, and cDNA was collected. The amplification reaction was carried out using the amplification primers mentioned above, and the amplification reaction products were collected.

[0021] The amplification reaction product was subjected to a single-base extension reaction using the single-base extension primers described above, and the single-base extension reaction product was collected.

[0022] The product of the single base extension reaction was mixed with a molecular weight internal standard and formamide, denatured, cooled, and then subjected to genotyping to obtain the SNP gene type of the sperm donor.

[0023] The present invention discloses the following technical effects:

[0024] The primer composition provided by this invention includes amplification primers with nucleotide sequences as shown in SEQ ID NO. 1-34 and single-base extension primers as shown in SEQ ID NO. 35-52, capable of amplifying 18 mRNA-cSNP markers specific to semen (stains). A SNaPshot complex detection kit is also provided. This invention further provides a method for identifying human semen samples, including extracting sample mRNA, reverse transcription to obtain cDNA, performing an amplification reaction using amplification primers, and then performing a single-base extension reaction using single-base extension primers. Experimental results show that the identification method of this invention can accurately identify individual semen donors, providing an effective technical method for directly associating semen (stain) types with their donors. This invention establishes a forensic semen (stain) identification kit with high consistency, good tissue specificity, and high sensitivity, providing an effective detection tool for semen (stain) identification, and is of great significance for achieving accurate semen (stain)-related criminal investigations and forensic identification. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 Capillary electrophoresis patterns of 18 semen-specific cSNP markers in a semen stain sample;

[0027] Figure 2 This is a graph showing the results of the typing consistency test.

[0028] Figure 3 This is a graph showing the sensitivity test results;

[0029] Figure 4 This is a graph showing the results of testing a mixture containing semen. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] The reaction thermal cycler of this invention is an ABI ProFlex PCR system, and the genetic analyzer is a 3500 series genetic analyzer; the molecular weight internal standard is GeneScan-LIZ 120 Standard with ORG fluorescent labeling; the fluorescence correction is performed using 5-DyeMatrix Standards.

[0036] Example 1

[0037] This embodiment provides a method for detecting mRNA-cSNP genetic markers for semen (spot) identification, including the following steps:

[0038] I. Screening of mRNAs and cSNP markers on mRNAs suitable for semen (stain) identification

[0039] This invention systematically searches published literature, PubMed database, Human Protein Atlas, and GnomAD database to screen for highly specific mRNA molecules expressed only in semen tissue. For the semen-specific mRNA molecules obtained through screening, cSNP markers are selected in the GnomAD database according to the following criteria: (1) the cSNP marker is located in the protein coding region of the specific gene; (2) the minor allele frequency (MAF) of each cSNP in the East Asian population is not less than 0.05; (3) the distance between the cSNP and the boundary of the adjacent exon is less than 500 bp; (4) primers with an amplicon length of less than 450 bp can be designed across introns.

[0040] Eighteen cSNP markers were ultimately identified from 16 sperm-specific mRNA genes. The specific mRNA-cSNP genetic markers are: PIWIL1-rs10848087, KLHL10-rs1529933, SPATA16-rs1515442, STK31-rs6945306, CALR3-rs9305079, SOX30-rs35793864, KLK2-rs198972, and TCP11. -rs2234045, TCP11-rs35693439, CATSPERZ-rs2286614, SORD-rs1042079, PRAC1-rs1054072, CKB-rs11 36165, PRM1-rs737008, GAPDHS-rs2239945, TPPP2-rs9624, SPATA22-rs1488689 and SPATA22-rs1488690.

[0041] II. Design of Amplification Primers and Single-Base Extension Primers

[0042] A SNaPshot complex detection kit containing multiplex amplification primers and multiplex single-base extension primers was developed, with ORG fluorescent labeling (GeneScan-LIZ 120Standard) used as the molecular weight internal standard.

[0043] The genetic markers and their corresponding amplification primer sequences in this embodiment are shown in Table 1, and the single-base extension primer sequences are shown in Table 2.

[0044] Table 1 Amplification Primer Sequences

[0045]

[0046] Table 2. Single-base extension primer sequences

[0047]

[0048]

[0049] III. Construction and Optimization of SNaPshot Composite Detection

[0050] The constructed SNaPshot composite detection system was optimized by adjusting reaction conditions such as the concentration ratio of amplification primers to single-base extension primers, the amount of cDNA template, the annealing temperature, and the number of cycles, so as to obtain balanced and stable single-base extension genotyping results and realize the composite detection of 18 mRNA-cSNP genetic markers.

[0051] The total amplification volume of the SNaPshot complex detection system containing 18 semen-specific mRNA-cSNP genetic markers in this embodiment is 15 μL, including 7.5 μL 2×Multiplex PCR Master Mix, 1.5 μL 5×Q-Solution, 3 μL H2O, 1.5 μL mixed amplification primers, and 1.5 μL cDNA; wherein the cDNA is obtained by reverse transcription of RNA extracted from semen (spots); the concentration of each primer is shown in Table 1.

[0052] PCR reactions were amplified on a PCR instrument. The multiplex PCR amplification program was as follows: 95℃ pre-denaturation for 10 min; 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 90 s, 35 cycles; 60℃ final extension for 60 min, and storage at 4℃.

[0053] The PCR product was purified using Exo I (14 U / μL) and shrimp alkaline phosphatase (rSAP). The total reaction volume was 10 μL, including 5 μL of PCR amplification product, 1.5 μL of Exo I, 2.5 μL of rSAP, and 1 μL of deionized water. The purification reaction procedure was as follows: reaction at 37°C for 2 h, heating at 80°C for 10 min, and storage at 4°C.

[0054] The purified PCR product was collected and subjected to a single-base extension reaction. The total volume of the single-base extension reaction system was 5 μL, including 2 μL of purified PCR product, 1 μL of mixed single-base extension primers, and 2 μL of SNaPshot Reaction Buffer; the concentrations of each primer are shown in Table 2.

[0055] The single-base extension reaction was performed on a PCR instrument. The reaction program was as follows: 96℃ pre-denaturation for 30s; 96℃ denaturation for 10s, 50℃ annealing for 5s, 60℃ extension for 30s, 35 cycles; 60℃ final extension for 1min, and storage at 4℃.

[0056] The monobasic extension reaction product was collected and purified. The total volume of the reaction system was 6 μL, including 5 μL of monobasic extension product and 1 μL of rSAP. The purification reaction procedure was as follows: reaction at 37 °C for 60 min, heating at 80 °C for 10 min, and storage at 4 °C.

[0057] IV. Establishment of Analytical Methods and Detection of Products

[0058] A genetic analyzer spectral correction (Matrix) file was established. During capillary electrophoresis, 1 μL of purified single-base extension reaction product was mixed with 8.5 μL of formamide and 0.5 μL of molecular weight internal standard (GeneScan-LIZ 120 Standard). The mixture was denatured at 95 °C for 3 min, followed by cooling on ice for 3 min. The genetic analyzer was used to perform genotyping of 18 mRNA-cSNP markers. Electrophoretic migration parameters of different alleles at each locus were obtained by capillary electrophoresis. Based on these parameters, corresponding Bin and Panel files were created according to the format requirements of GeneMapperID-X v1.6 software to establish the electrophoretic analysis method.

[0059] The genotyping profiles of semen cDNA samples obtained using the above method are as follows: Figure 1 As shown.

[0060] Example 2

[0061] This embodiment describes the detection of semen cDNA in 50 samples using the method provided in Example 1.

[0062] Semen samples were collected from unrelated individuals, and RNA was extracted, quantified, and reverse transcribed to prepare cDNA samples. The cDNA samples were then subjected to complex detection using 18 mRNA-cSNP markers according to the system and kit constructed in Example 1. All samples showed effective amplification of the cSNP markers and successful genotyping. The calculated total discrimination power (TDP) was 0.99994.

[0063] Example 3

[0064] This embodiment provides forensic verification of the method provided in Embodiment 1. The specific experiments and results are as follows:

[0065] In accordance with the requirements of the Scientific Working Group for DNA Analysis Methods (SWGDAM), the consistency, sensitivity, and tissue specificity of the SNaPshot composite detection system and its kit constructed in Example 1 were studied.

[0066] Forensic verification results showed that the system and kit constructed in Example 1 exhibited high consistency in the detection results of semen (stains) in both cDNA and genomic DNA. Figure 2 It exhibits good tissue specificity and high sensitivity, successfully detecting samples after reverse transcription of RNA up to 1.25 ng. Figure 3 It can detect semen components in different mixed samples, such as semen and saliva, semen and vaginal fluid, semen and venous blood, and semen and menstrual blood. Figure 4 ), Figure 4 The average number of peaks and average peak height of semen were determined in a mixture of four bodily fluid components: saliva, menstrual blood, vaginal secretions, and semen. The genotypes of 18 cSNPs of semen components in each mixture containing 50 μL of semen were successfully obtained.

[0067] As can be seen from the above embodiments, the primer composition, kit and method for detecting semen-specific mRNA-cSNP markers constructed by the present invention based on SNaPshot technology can be applied to determine whether biological samples contain semen (spots) and provide SNP gene typing of semen donors, providing a brand-new detection method for forensic semen (spot) identification.

[0068] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a primer composition in the preparation of products for detecting semen-specific mRNA-cSNP markers, characterized in that, Including amplification primers and single-base extension primers; The amplification primers are shown in Table 1: Table 1 ； The single-base extension primers are shown in Table 2: Table 2 。 2. The application as described in claim 1, characterized in that, The product in question is a reagent kit.

3. A kit for detecting semen-specific mRNA-cSNP markers, characterized in that, Includes the primer composition described in claim 1.

4. The kit according to claim 3, characterized in that, It also includes amplification reaction reagents and single-base extension reaction reagents.

5. The use of a primer composition as described in claim 1 or a kit as described in claim 3 or 4 in the preparation of a product for identifying semen, characterized in that, The identification of semen includes SNP genotyping of the semen donor.

6. The use of a primer composition as described in claim 1 or a kit as described in claim 3 or 4 in the identification of semen, characterized in that, The identification of semen includes SNP genotyping of the semen donor.

7. A method for obtaining SNP genotyping of sperm donors, characterized in that, Includes the following steps: mRNA is extracted from the sample to be tested, reverse transcribed, cDNA is collected, amplification reaction is performed using the amplification primers described in claim 1, and the amplification reaction products are collected. The amplification reaction product was subjected to a single-base extension reaction using the single-base extension primers described in claim 1, and the single-base extension reaction product was collected. The product of the single base extension reaction was mixed with a molecular weight internal standard and formamide, denatured, cooled, and then subjected to genotyping to obtain the SNP gene type of the sperm donor.

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