A cSNP primer composition, kit and application thereof for body fluid tracing based on second-generation sequencing technology

Abstract

This invention discloses a cSNP primer composition, kit, and its application for body fluid tracing based on next-generation sequencing technology, relating to the fields of forensic biological evidence and molecular identification technology. The cSNP primer composition includes primer pairs amplifying 34 cSNP genetic markers. The primer composition for body fluid tracing based on next-generation sequencing technology provided by this invention relates to 34 cSNP markers for identifying five types of body fluids. This invention has developed and established a kit capable of determining the body fluid type and genotyping the body fluid donor cSNPs. Using the primer composition or kit provided by this invention, highly consistent genotyping results can be obtained, with high sensitivity and good tissue specificity. It provides an effective detection tool for directly associating common body fluid types and body fluid donors, and is of great significance for further research on body fluid identification and achieving accurate and efficient forensic identification related to body fluids.

Description

Technical Field

[0001] This invention relates to the fields of forensic biological evidence and molecular identification technology, and in particular to a cSNP primer composition, kit, and application for tracing body fluids based on next-generation sequencing technology. Background Technology

[0002] In the field of forensic medicine, there are many methods for identifying body fluid types. Biochemical and serological methods, enzyme activity detection, and cytological examination are commonly used for estimating and confirming the type of body fluid, and most have advantages such as simplicity and speed. However, there are still many limitations in practical application. First, each experiment can only test one type of body fluid. When dealing with multiple body fluids, the testing procedure is cumbersome and requires a significant investment of time and manpower. Second, the specificity and sensitivity of the tests vary, and they are easily affected by the integrity of the sample itself and interfering substances, leading to false negatives or false positives. In addition, traditional identification methods consume large amounts of sample and can damage the sample, which is extremely disadvantageous for small quantities of biological samples. Moreover, the interpretation of results from these traditional methods is often susceptible to subjective influence, with a risk of misinterpretation.

[0003] To overcome the limitations of traditional detection methods and solve forensic body fluid identification problems more accurately and efficiently, researchers have proposed several new methods in recent years, including DNA methylation pattern analysis, microbial diversity analysis, and RNA expression pattern analysis. Among these, mRNA analysis, due to its mature analytical techniques and high specificity, has been widely validated for its ability to identify common body fluids and tissues such as blood, semen, saliva, vaginal secretions, menstrual blood, and skin. Several European laboratories have also explored and validated methods for interpreting mRNA analysis results for body fluid identification through collaborative experiments. For example, the European DNA Working Group (EDNAP) and the Forensic RNA Profiling Group (FoRNAP) systematically evaluated the identification efficacy of mRNA analysis in various samples using capillary electrophoresis (CE) and massively parallel sequencing (MPS) techniques, respectively. These research results indicate that applying mRNA analysis for body fluid identification can serve as an alternative strategy to traditional body fluid identification methods.

[0004] Generally, body fluid identification uses body fluid-specific mRNA to determine the body fluid type and DNA genotyping results from the same sample for individual identification. However, in mixed plaques, DNA STR typing results and body fluid-specific mRNA results are independent of each other and lack a direct link. It is impossible to determine which body fluid the STR typing results originate from, and direct application may erroneously correlate DNA evidence with RNA evidence.

[0005] Using coding region single nucleotide polymorphisms (cSNPs) on body fluid-specific mRNAs can directly link specific body fluids in mixed plaques to their donors, avoiding association fallacies. This theory was first proposed by Hanson et al. in 2015, who experimentally demonstrated the feasibility of assigning body fluids to specific individuals using body fluid-specific cSNPs and initially constructed a corresponding detection method. In recent years, more research teams have attempted to combine mRNA and cSNPs on different platforms to construct body fluid identification systems, aiming to obtain genetic polymorphism information of the individuals from whom the body fluids originated while determining the body fluid type. These studies demonstrate the significant application potential of cSNP markers in the identification of body fluids (plaques) and mixed body fluids (plaques). However, the number of currently recognized body fluid-specific mRNAs is relatively small, and the number of cSNPs is limited. Even with multiple markers combined, it is difficult to obtain individual identification capabilities that match conventional STR typing. Furthermore, SNPs exhibit significant population differences, and currently reported cSNPs are more suitable for body fluid tracing in European populations. Therefore, it is necessary to find cSNPs that are more suitable for the Chinese population and have high fluid specificity to supplement and improve the fluid traceability site database.

[0006] Existing SNP detection methods include microarrays, TaqMan probe assays, SNaPshot assays, mass spectrometry in flight, and next-generation sequencing. Microarray technology can detect known SNP genotypes with high throughput, but it is prone to false positives and is generally used for site screening. TaqMan probe assays can detect SNPs with high specificity, but the number of sites detected at a time is limited, making it unsuitable for multi-site complex analysis. SNaPshot assays are highly sensitive and can be performed on capillary electrophoresis platforms, but the number of sites detectable in a single-base extension reaction system is limited, and the experimental steps are cumbersome, requiring multiple product transfers, which can easily lead to loss and contamination. This invention aims to develop a new detection method for tracing the origins of multiple cSNP genetic markers in multiple samples using next-generation sequencing technology. Summary of the Invention

[0007] The purpose of this invention is to provide a cSNP primer composition, kit, and application for body fluid tracing based on next-generation sequencing technology, thereby addressing the problems existing in the prior art. This primer composition can detect highly consistent genotyping results with high sensitivity and good tissue specificity, providing an effective detection tool for directly associating common body fluid types and donors. This is of great significance for further research on body fluid identification and for achieving accurate and efficient forensic identification related to body fluids.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] This invention provides a cSNP primer composition for tracing the origin of cerebral nucleotides in bodily fluids based on next-generation sequencing technology, comprising primer pairs for amplifying 34 cSNP genetic markers as shown below:

[0010]

[0011]

[0012]

[0013] Each primer pair was grouped according to primer type, and PCR amplification was performed in two systems, T1 and T2, respectively.

[0014] The present invention also provides the use of the above-described cSNP primer composition in the preparation of a kit for tracing body fluids.

[0015] The present invention also provides a kit for tracing the origin of bodily fluids, comprising the above-described cSNP primer composition.

[0016] Furthermore, the kit also includes library construction reagents and sequencing reagents.

[0017] The present invention also provides the application of the above-described cSNP primer composition or kit in body fluid tracing.

[0018] Furthermore, the types of bodily fluids include semen, saliva, vaginal secretions, menstrual blood, and venous blood.

[0019] This invention also provides a method for identifying body fluid types or differentiating body fluid donors using cSNP genetic markers, comprising the following steps:

[0020] RNA was extracted from the sample to be tested and reverse transcribed into cDNA;

[0021] The cSNP primer composition described above is used to construct a library and sequence the cDNA of the sample to be tested. The obtained sequencing data is analyzed to obtain cSNP typing and to determine the body fluid type of the sample or to distinguish the body fluid donor.

[0022] Furthermore, the types of bodily fluids include semen, saliva, vaginal secretions, menstrual blood, and venous blood.

[0023] Furthermore, the library construction includes a first round of PCR reaction and a second round of adapter reaction;

[0024] The system for the first round of PCR reaction was: 3.5 μL Enhancer buffer NB, 2.5 μL Enhancer buffer M, 10 μL IGT-EM808 polymerase mixture, 8 μL deionized water, 5 μL primer mixture and 1 μL cDNA;

[0025] The second round of adapter reaction consisted of: 2.5 μL Enhancer buffer M, 10 μL IGT-EM808 polymerase mixture, 2 μL deionized water, 2 μL adapter primer mixture, and 13.5 μL first round PCR product.

[0026] Furthermore, the procedure for the first round of PCR reaction was as follows: preheating at 105℃; pre-denaturation at 95℃ for 3 minutes and 30 seconds, denaturation at 98℃ for 20 seconds, annealing at 60℃ for 3 minutes, extension at 72℃ for 5 minutes, 22 cycles; final extension at 72℃ for 5 minutes.

[0027] The procedure for the second round of joint reaction is as follows: preheating at 105°C; pre-denaturation at 95°C for 3 minutes and 30 seconds, denaturation at 98°C for 20 seconds, annealing at 58°C for 1 minute, extension at 72°C for 30 seconds, 9 cycles; final extension at 72°C for 5 minutes.

[0028] The present invention discloses the following technical effects:

[0029] This invention provides a primer composition for tracing bodily fluids based on next-generation sequencing technology. This composition involves 34 specific cSNP markers for identifying five bodily fluids (semen, saliva, vaginal secretions, menstrual blood, and venous blood). This invention also develops and establishes a kit for determining the type of these five bodily fluids and genotyping the cSNPs of bodily fluid donors. Using the primer composition or kit provided by this invention, highly consistent genotyping results can be obtained with high sensitivity and good tissue specificity. This provides an effective detection tool for directly associating common bodily fluid types and donors, and is of great significance for further research on bodily fluid identification and for achieving accurate and efficient forensic identification related to bodily fluids. Attached Figure Description

[0030] 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.

[0031] Figure 1 The distribution of average sequencing depth (reads) of cSNP markers specific to various bodily fluids is shown; a, e represent blood, menstrual blood, saliva, vaginal secretions, and semen samples, respectively. Detailed Implementation

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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 readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] 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.

[0037] Example 1

[0038] Thirty-four cSNP genetic markers on 17 fluid-specific mRNA molecules were screened from five common bodily fluids (semen, saliva, vaginal secretions, menstrual blood, and venous blood). The minor allele frequency (MAF) of all these markers in East Asian populations was greater than or equal to 0.05 (details are shown in Table 1). Primers were designed based on these 34 cSNP markers (details are shown in Table 2). Each primer was used in two systems (T1 and T2), and multiplex PCR amplification was performed separately for each system during detection.

[0039] Table 1. Basic Information on cSNP Genetic Markers

[0040]

[0041]

[0042] Note: SA, SE, VB, VS, and MB refer to saliva, semen, venous blood, vaginal secretions, and menstrual blood, respectively. Screening criteria for the five humor-specific mRNAs and cSNPs: mRNAs with humor-specificity verified in literature, research, and databases; SNPs located in autosomal coding regions (i.e., cSNPs); cSNP MAF greater than or equal to 0.05; cSNP distance from the nearest intron within 400 bp.

[0043] Table 2 Primer Sequence Details

[0044]

[0045]

[0046]

[0047]

[0048] Note: 1. T1 and T2 represent different PCR reaction systems. 2. rs946696 and rs3737277 were simultaneously amplified using primers shown in SEQ ID NO. 11-12; rs2075739 and rs1977734 were simultaneously amplified using primers shown in SEQ ID NO. 45-46; rs3094672 and rs2517554 were simultaneously amplified using primers shown in SEQ ID NO. 63-64; rs4713420 and rs12179536 were simultaneously amplified using primers shown in SEQ ID NO. 67-68; rs76504934 was amplified first to a long fragment using primers shown in SEQ ID NO. 39-40, and then amplified to a short fragment using primers shown in SEQ ID NO. 41-42; rs2286614 was amplified first to a long fragment using primers shown in SEQ ID NO. 47-48, and then amplified to a short fragment using primers shown in SEQ ID NO. 41-42. Primers NO.49-50 amplified a short fragment; rs198977 was amplified to a long fragment first using primers NO.51-52, and then to a short fragment using primers NO.53-54; rs198972 was amplified to a long fragment first using primers NO.55-56, and then to a short fragment using primers NO.57-58. 3. Based on the flanking sequences of the screened cSNPs, multiplex PCR primers were designed using primer design software. The design principles included: 1) At least one of the front and back primers should span an intron, or the two primers should be located in different exons to ensure that the amplicon is a cDNA-specific product and avoid gDNA contamination; 2) The primer length should be 18-30 bp, and the primer ends should retain at least 5 bp from the cSNP location; 3) The annealing temperature (Tm value) should be 59-61℃; the GC content should be 40-60%; there should be no hairpin structures, primer dimers, or mismatches; 4) The product length should not exceed 450 bp; 5) There should be no other valid amplification products in the specificity detection results of the NCBI Primer-BLAST database.

[0049] This embodiment provides a method for tracing the origin of bodily fluids based on next-generation sequencing technology, including the following steps:

[0050] (1) Extract RNA from the sample to be tested and reverse transcribe it into cDNA;

[0051] (2) Use The first round of multiplex amplification was performed using the Library Prep Kit, and the reaction system is shown in Table 3.

[0052] Table 3. Library Construction First-Round PCR Reaction System

[0053]

[0054] Note: When constructing the library, Primerpool T1 / T2 is a mixture of two primers from SEQ ID NO.1 to 68 in Table 2, and the concentration of each primer is shown in Table 2.

[0055] PCR reactions were performed on a 9700 or other PCR instrument. The multiplex PCR reaction program was as follows: preheating at 105°C; pre-denaturation at 95°C for 3 minutes and 30 seconds, denaturation at 98°C for 20 seconds, annealing at 60°C for 3 minutes, extension at 72°C for 5 minutes, for 22 cycles; final extension at 72°C for 5 minutes; after the reaction, 15 μL of amplification product was taken from each of the T1 and T2 reaction tubes and mixed in equal volumes.

[0056] (3) First round of magnetic bead purification products

[0057] Using IGT TM Pure Beads are used to purify first-round PCR products. Before use, the beads need to be equilibrated at room temperature for 30 minutes and then resuspended.

[0058] (4) The second round of adapter sequence ligation PCR reaction, the reaction system is shown in Table 4.

[0059] Table 4. Joint Connection Reaction System

[0060]

[0061] Note: Connector primer sequence: 5'-AATGATACGGCGACCACCGAGATCTACAC-i5 Index-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-Insert-TGGAATTCTCTCGGGTGCCAAGGAACTCCA GTCAC-i7Index'-ATCTCGTATGCCGTCTTCTGCTTG-3'.

[0062] PCR reactions were amplified on a 9700 or other PCR instrument. The reaction program for multiplex PCR was as follows: preheating at 105°C; pre-denaturation at 95°C for 3 minutes and 30 seconds, denaturation at 98°C for 20 seconds, annealing at 58°C for 1 minute, extension at 72°C for 30 seconds, for 9 cycles; final extension at 72°C for 5 minutes.

[0063] (5) Second round of magnetic bead purification products

[0064] Using IGT TM Pure Beads are used to purify second-round PCR products. Before use, the beads need to be equilibrated at room temperature for 30 minutes and then resuspended.

[0065] (6) Purification and quantification of libraries

[0066] Library quantification and quality control were performed using a Qubit 2.0 fluorescence quantitative analyzer and a Qsep 400 fully automated nucleic acid and protein analysis system.

[0067] (7) Sequencing and Data Analysis

[0068] Sequencing was performed on an Illumina NOVASEQ 6000 platform using the MiSeq Reagent Kit V2. After obtaining the data, sequencing adapters, low-quality bases, or N-base sequences were removed. The sequences were then compared and ordered with the human reference genome (GRCh37 / hg19) using BWA software to obtain SAM result files. The SAM files were converted to BAM format using Samtools, and library quality was assessed. Genotyping was then performed using GATK-3.8.0 and Varscan-v2.4.3. Sequencing depth was calculated for each locus, and the percentage of sequencing depth for each humor-specific locus was determined. The cSNP genotyping results were then summarized.

[0069] Example 2

[0070] The method provided in Example 1 was used to detect cDNA in 118 samples of semen, saliva, vaginal secretions, menstrual blood, and venous blood.

[0071] Semen, saliva, vaginal secretions, menstrual blood, and venous blood samples were collected from unrelated individuals. RNA was extracted, quantified, and reverse transcribed to prepare cDNA samples. The cDNA samples were then used to construct libraries containing 34 cSNP markers, sequenced, and analyzed using the method described in Example 1. The average sequencing depth (reads) distribution of each fluid-specific cSNP marker in blood, menstrual blood, saliva, vaginal secretions, and semen samples is shown in the figure. Figure 1 The sequencing quality is shown in Table 5.

[0072] Table 5 Summary of sequencing data for various bodily fluid samples

[0073]

[0074] Note: SA, SE, VB, VS and MB refer to saliva, semen, venous blood, vaginal secretions and menstrual blood, respectively.

[0075] Example 3

[0076] Using the method of Example 1, saliva and venous blood from nine common non-human samples (goose, donkey, chicken, sheep, pig, cow, dog, cat, and pigeon) were tested, and no specific products were detected, indicating that the method of the present invention for determining body fluid type has strong species specificity.

[0077] Example 4

[0078] A kit for tracing the origin of bodily fluids includes the cSNP primer composition, library construction reagents, and sequencing reagents shown in Table 2.

[0079] Example 5

[0080] This embodiment uses the method provided in Example 1 to detect a mixture of two bodily fluids. The specific experiments and results are as follows:

[0081] Mixed samples of two bodily fluids were prepared, including semen and saliva, semen and vaginal secretions, semen and blood, and blood and menstrual blood. Additionally, mixed samples of three bodily fluids (semen, menstrual blood, and blood) were prepared. The cDNA libraries of each sample were constructed, sequenced, and analyzed using the method provided in Example 1. The genotyping results are shown in Tables 6 and 7.

[0082] Table 6. Classification of Two-Component Simulated Mixture Samples and Reference Samples

[0083]

[0084]

[0085]

[0086]

[0087] Note: SA, SE, VB, VS, and MB refer to saliva, semen, venous blood, vaginal secretions, and menstrual blood samples, respectively. The simulated mixture is prepared by mixing the above RNA components in a 1:1 ratio. A negative cSNP genotyping result is indicated by "-".

[0088] Table 7. Classification of Three-Component Simulated Mixture Samples and Reference Samples

[0089]

[0090] Note: SA, SE, VB, VS and MB refer to saliva, semen, venous blood, vaginal secretions and menstrual blood samples, respectively. The simulated mixture is made by mixing the above RNA components in a 1:1:1 ratio.

[0091] Methods for determining the type of bodily fluid sample:

[0092] After obtaining valid data through cSNP targeted sequencing of each bodily fluid sample, the bodily fluid type was determined based on the detection of bodily fluid-specific markers (the percentage of a single marker reads in the total sample reads > 0.1%). When half or more of the specific markers for semen, blood, vaginal secretions, or menstrual blood were detected in the mixed sample, the sample was considered to contain semen, blood, vaginal secretions, or menstrual blood components. When half or more of the specific markers for saliva were detected, and the sample included the saliva-specific marker (rs10845341), the mixed patch was considered to contain saliva components. After determining the bodily fluid components based on the detection of various markers in the mixed bodily fluid sample, the genotypes of the bodily fluid-specific cSNP markers in the mixed patch were compared with the corresponding genotypes of the reference individual's DNA to identify the bodily fluid donor. If the genotype matched the reference individual, the bodily fluid component was considered to originate from that donor; if there was one or more discrepancies, the donor could be excluded. As shown in Table 6, the saliva-specific marker typing in the saliva and semen mixture was completely consistent with the reference sample of donor 1, but inconsistent with donor 2 at 3 / 6 sites, supporting that the saliva came from donor 1 (saliva donor); the semen-specific marker typing in the mixed stain was consistent with donor 2, but inconsistent with donor 1 at 3 / 3 sites, supporting that the semen came from donor 2 (sperm donor).

[0093] As can be seen from the above embodiments, the primer composition and kit for detecting body fluid-specific cSNP markers based on next-generation sequencing technology provided by the present invention can be applied to determine the body fluid type of five body fluids and can provide cSNP gene typing results of body fluid donors, providing a new detection method for forensic body fluid identification.

[0094] 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 cSNP primer composition in the preparation of a kit for tracing the origin of bodily fluids, characterized in that, The type of bodily fluid is semen, saliva, vaginal secretions, menstrual blood, or venous blood; The cSNP primer composition consists of primer pairs that amplify 34 cSNP genetic markers, as shown below: Each primer pair was grouped according to primer type, and PCR amplification was performed in two systems, T1 and T2, respectively.

2. An application of a reagent kit in body fluid traceability, characterized in that, The type of bodily fluid is semen, saliva, vaginal secretions, menstrual blood, or venous blood; The kit includes the cSNP primer composition as described in claim 1.

3. The application according to claim 2, characterized in that, The kit also includes library construction reagents and sequencing reagents.

4. The application of the cSNP primer composition as described in claim 1 in body fluid tracing, characterized in that, The types of bodily fluids mentioned are semen, saliva, vaginal secretions, menstrual blood, or venous blood.

5. A method for identifying body fluid types or differentiating body fluid donors using cSNP genetic markers, characterized in that, Includes the following steps: RNA was extracted from the sample to be tested and reverse transcribed into cDNA; The cSNP primer composition described in claim 1 is used to construct a library and sequence the cDNA of the sample to be tested. The obtained sequencing data is analyzed to obtain cSNP typing and to determine the body fluid type of the sample to be tested or to distinguish the body fluid donor. The types of bodily fluids mentioned are semen, saliva, vaginal secretions, menstrual blood, or venous blood.

6. The method according to claim 5, characterized in that, The library construction includes a first round of PCR reaction and a second round of adapter reaction; The system for the first round of PCR reaction was: 3.5 μL Enhancer buffer NB, 2.5 μL Enhancer buffer M, 10 μL IGT-EM808 polymerase mixture, 8 μL deionized water, 5 μL primer mixture and 1 μL cDNA; The second round of adapter reaction consisted of: 2.5 μL Enhancer buffer M, 10 μL IGT-EM808 polymerase mixture, 2 μL deionized water, 2 μL adapter primer mixture, and 13.5 μL first round PCR product.

7. The method according to claim 6, characterized in that, The procedure for the first round of PCR reaction was as follows: preheating at 105℃; pre-denaturation at 95℃ for 3 minutes and 30 seconds, denaturation at 98℃ for 20 seconds, annealing at 60℃ for 3 minutes, extension at 72℃ for 5 minutes, 22 cycles; final extension at 72℃ for 5 minutes. The procedure for the second round of joint reaction is as follows: preheating at 105°C; pre-denaturation at 95°C for 3 minutes and 30 seconds, denaturation at 98°C for 20 seconds, annealing at 58°C for 1 minute, extension at 72°C for 30 seconds, 9 cycles; final extension at 72°C for 5 minutes.

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