A primer combination, a kit and a method for identifying an mRNA / cInDel genetic marker for body fluid identification

Abstract

The application discloses a primer combination, a kit and an identification method of mRNA / cInDel genetic markers for body fluid identification, and relates to the fields of forensic material evidence and molecular identification technology. The primer combination comprises primer pairs for amplifying 19 genetic markers SLC4A1, ALAS2, STATH, HTN3, KRT4, PRM1, TCP11, SEMG1, ODF1, MMP11, STC1, LPAR3, COL6A3, CYP2B7P1, GPAT3, Lcris, FAM83D, RPS4Y1 and XIST. The primer combination can be used to identify the types of body fluids (semen, saliva, vaginal fluid, menstrual blood and venous blood) and perform InDel genotyping on the donors of the body fluids, and has the characteristics of high consistency, good tissue specificity, high sensitivity and strong species specificity, and provides a theoretical basis and a detection method for body fluid tracing.

Description

Technical Field

[0001] This invention relates to the fields of forensic biological evidence and molecular identification technology, and in particular to a primer combination, kit, and identification method for mRNA / cInDel genetic markers used for body fluid identification. Background Technology

[0002] In the field of forensic medicine, there are many methods for identifying body fluid types. While traditional biochemical or immunological analyses are relatively simple and rapid, they also have some insurmountable limitations. First, cross-reactivity exists between some body fluids and tissues, resulting in low detection specificity for these methods. Second, traditional identification methods can damage the sample, which is extremely disadvantageous for small quantities of biological samples. Furthermore, the interpretation of results from traditional methods is susceptible to human error, potentially leading to misinterpretations. Finally, these traditional methods can only detect a very limited number of body fluid types. Due to these limitations, researchers have recently proposed new methods for body fluid identification to more accurately address related issues in forensic medicine, including DNA methylation pattern analysis, copy number variation analysis, microbial characterization analysis, and RNA analysis. Among these new methods, RNA analysis has gradually become one of the more mature methods for body fluid identification due to the rapid development of transcriptome analysis technology. For several common bodily fluids or tissues (such as blood, semen, saliva, vaginal fluid, menstrual blood, and skin), a series of mRNA-specific markers have been widely validated, and international laboratories have recommended methods for interpreting mRNA analysis results for bodily fluid identification. Since 2011, the European DNA Working Group (EDNAP) and the Forensic RNA Profiling Group (FoRNAP) have systematically evaluated the identification efficacy of mRNA analysis in various samples for bodily fluid identification using capillary electrophoresis (CE) or massively parallel sequencing (MPS) techniques. These results demonstrate the promising application prospects of mRNA analysis for bodily fluid identification.

[0003] When performing body fluid identification, it is usually necessary to extract mRNA and DNA from the same sample and test them separately. The mRNA-specific results are used to determine the body fluid type, while the STR typing results in the DNA are used for individual identification. However, in mixed sample identification, the correlation between STR typing results and mRNA-specific results is not direct, because STR typing results may come from body fluids or tissues that were not identified in the mRNA analysis, thus causing erroneous association between DNA evidence and RNA evidence.

[0004] To find more suitable methods for identifying body fluids (spots) and mixed body fluids (spots), Hanson et al. first proposed in 2015 the feasibility of using coding region single nucleotide polymorphisms (cSNPs) in body fluid-specific mRNAs to assign body fluids to specific individuals. This directly links body fluid type and donor polymorphism information, and they attempted to construct corresponding detection methods. In recently published studies, some research teams have also attempted to incorporate some cSNP markers into existing mRNA body fluid identification detection systems, obtaining genetic polymorphism information of the relevant body fluid source individuals while determining body fluid type. Although the number of cSNPs currently available for body fluid identification is insufficient to achieve the same satisfactory individual identification capability as conventional STR typing, these studies demonstrate the enormous application potential of cSNP markers in identifying body fluids (spots) and mixed body fluids (spots).

[0005] However, the detection methods for SNPs, including TaqMan probe method, SNaPshot method, mass spectrometry of flight, or MPS method, are not widely used in forensic biological evidence laboratories, which hinders the application and popularization of cSNP body fluid identification methods. Currently, there is an urgent need to develop a new body fluid identification method that is highly specific, highly sensitive, simple and easy to operate, and readily applicable in forensic biological evidence laboratories. Summary of the Invention

[0006] The purpose of this invention is to provide a primer combination, kit, and identification method for mRNA / cInDel genetic markers used for body fluid identification, in order to solve the problems existing in the prior art. Using this primer combination, it is possible to determine the type of body fluid (semen, saliva, vaginal fluid, menstrual blood, and venous blood) and to genotype the InDel gene of body fluid donors. It has the characteristics of high consistency, good tissue specificity, high sensitivity, and strong species specificity. This invention provides a theoretical basis and detection method for body fluid tracing.

[0007] InDel (Insertion / Deltion) genetic markers, as a special type of SNP marker, are similar to SNPs in that they have the advantages of low mutation rate and short amplicon length. Another prominent feature of InDel is that its genotype can be determined based on DNA fragment length polymorphism, thus enabling genotyping using conventional PCR-CE techniques. This experimental procedure is simple and easy to perform, making it readily applicable in forensic biological evidence laboratories. Therefore, theoretically, InDel (cInDel) markers in the coding regions of body fluid-specific mRNAs can be integrated into existing mRNA analysis systems for determining body fluid types and genotyping of InDel donors.

[0008] Based on the above research and development approach, the present invention provides the following solution:

[0009] This invention provides a primer combination for mRNA / cInDel genetic markers for body fluid identification, comprising primer pairs for amplifying 19 genetic markers as shown below:

[0010]

[0011]

[0012] Furthermore, in the primer pairs amplifying the 19 genetic markers, at least one primer in each primer pair is labeled with a fluorescent dye, which is selected from FAM, HEX, TRMRA and ROX.

[0013] Furthermore, SLC4A1, ALAS2, STATH, KRT4, ODF1, and COL6A3 are labeled with FAM; HTN3, LPAR3, CYP2B7P1, GPAT3, Lcris, and FAM83D are labeled with HEX; PRM1, SEMG1, MMP11, and RPS4Y1 are labeled with TAMRA; and TCP11, STC1, and XIST are labeled with ROX.

[0014] The present invention also provides the application of the above-described primer combinations in the preparation of kits for the identification of body fluids.

[0015] The present invention also provides a kit for identifying body fluids, comprising the primer combination described above.

[0016] Furthermore, the kit also includes 2×MultiplexPCRMasterMix, 5×Q-Solution, and deionized water.

[0017] This invention also provides a method for identifying bodily fluids, comprising the following steps:

[0018] RNA was extracted from the body fluids to be tested and reverse transcribed into cDNA;

[0019] Using the cDNA as a template, PCR amplification was performed using the primer combination described above to obtain the amplification product;

[0020] The amplification products are used for typing to determine the body fluid type of the tested fluid.

[0021] Furthermore, the PCR amplification reaction system includes: 7.5 μL 2×PCRMasterMix, 1.5 μL 5×Q-Solution, 3 μL deionized water, 1.5 μL primer mixture, and 1.5 μL cDNA.

[0022] Furthermore, the PCR amplification reaction program includes: 95℃ pre-denaturation for 15 minutes; 94℃ denaturation for 30 seconds, 60℃ annealing for 1 minute and 30 seconds, 72℃ extension for 1 minute and 30 seconds, 35 cycles; and 60℃ final extension for 60 minutes.

[0023] The present invention also provides the application of the above-described primer combinations or kits in the identification of body fluids.

[0024] The present invention discloses the following technical effects:

[0025] This invention can amplify 19 mRNA / cInDel specific markers for 5 bodily fluids (semen, saliva, vaginal fluid, menstrual blood, and venous blood), and provides a kit containing the above-mentioned fluorescently modified genetic marker multiplex amplification system.

[0026] This invention provides an effective method for further research on body fluid identification and for genotyping that directly links body fluid types and body fluid donors.

[0027] This invention establishes a forensic body fluid identification kit with high consistency, good tissue specificity, high sensitivity, and strong species specificity. It provides an effective detection tool for the identification of five common body fluids (semen, saliva, vaginal fluid, menstrual blood, and venous blood), which is of great significance for achieving accurate forensic identification of body fluids. Attached Figure Description

[0028] 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.

[0029] Figure 1 Capillary electrophoresis patterns of five bodily fluids; where SE represents semen; VB represents venous blood; SA represents saliva; MB represents menstrual blood; and VS represents vaginal fluid.

[0030] Figure 2 These are the results of a semen sensitivity test;

[0031] Figure 3 The results are from a venous blood sensitivity test.

[0032] Figure 4 These are the results of a saliva sensitivity test;

[0033] Figure 5 These are the results of a vaginal fluid sensitivity test.

[0034] Figure 6 The results are from a menstrual blood sensitivity test.

[0035] Figure 7 Capillary electrophoresis pattern of a mixed sample of saliva and semen;

[0036] Figure 8 This is a capillary electrophoresis pattern of a mixed sample of vaginal fluid and semen. Detailed Implementation

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] Example 1

[0043] This embodiment provides a method for using mRNA / cInDel genetic markers for the identification of five bodily fluids, comprising the following steps:

[0044] I. Screening of mRNAs suitable for body fluid identification and cInDel markers on mRNAs

[0045] This invention, referencing literature reports and the GnomAD database, screened five humor-specific mRNAs and cInDel. The screening criteria included: mRNAs verified to be humor-specific; InDel located in an autosomal coding region (i.e., cInDel); a minor allele frequency (MAF) greater than or equal to 0.05 for cInDel; cInDel allele length varying between 1-30 bp; and the distance between cInDel and its nearest intron within 400 bp.

[0046] Ultimately, 19 humor-specific mRNA / cInDel genetic markers were screened: SLC4A1, ALAS2, STATH, HTN3, KRT4 (rs11267392), PRM1, TCP11 (rs142993601+rs142118879), SEMG1, ODF1 (rs752086061+rs757727788), MMP11, STC1, LPAR3 (rs58203951), COL6A3 (rs35879189), CYP2B7P1, GPAT3 (rs35602893), Lcris, FAM83D (rs534813259), RPS4Y1, and XIST. Among them, ODF1 (r s752086061+rs757727788), TCP11(rs142993601+rs142118879), PRM1 and SEMG1 are semen-specific markers; KRT4(rs11267392), STATH and HTN3 are saliva-specific markers; GPAT3(rs35602893), CYP2B7P1, Lcris and FAM83D(rs534813259) are vaginal fluid-specific markers; COL6A3(rs35879189), STC1, LPAR3(rs58203951) and MMP11 are menstrual blood-specific markers; SLC4A1 and ALAS2 are venous blood-specific markers; XIST and RPS4Y1 are sex markers.

[0047] II. Amplification Primer Design

[0048] A multiplex amplification detection kit containing five fluorescent labels (FAM, HEX, TAMRA, ROX, and ORG) was developed, with ORG fluorescent labeling (T-500) serving as the molecular weight internal standard.

[0049] The mRNA / cInDel genetic markers, their corresponding amplification primer sequences, and the marker fluorescence of this embodiment are shown in Table 1.

[0050] Table 1 Primer sequences and fluorescent labels used for multiplex amplification.

[0051]

[0052] III. Construction and Optimization of Multiplex PCR Amplification Systems

[0053] The constructed multiplex amplification system was adjusted and optimized, including PCR reaction conditions such as primer concentration, cDNA template amount, annealing temperature, and cycle number, to obtain balanced and stable PCR product typing results, achieving multiplex amplification of 19 mRNA / cInDel genetic markers.

[0054] This embodiment uses a 15 μL multiplex PCR amplification system for 19 mRNA / cInDel genetic markers specific to five body fluids. The system includes 7.5 μL of 2×Multiplex PCR Master Mix, 1.5 μL of 5×Q-Solution, 3 μL of H2O, 1.5 μL of primer mixture, and 1.5 μL of cDNA. The cDNA was obtained by reverse transcription of RNA extracted from semen, saliva, vaginal fluid, menstrual blood, and venous blood.

[0055] PCR reactions were amplified on a 9700 or other PCR instrument. The multiplex PCR reaction program was as follows: 95℃ pre-denaturation for 15 minutes; 94℃ denaturation for 30 seconds, 60℃ annealing for 1 minute and 30 seconds, 72℃ extension for 1 minute and 30 seconds, 35 cycles; 60℃ final extension for 60 minutes; and storage at 4℃.

[0056] IV. Establishment of Analytical Methods and Detection of Multiplex Amplification Products

[0057] Establish spectral correction (Matrix) files for the 3100 series, 3130 series, or 3500 series genetic analyzers. During capillary electrophoresis, mix 1 μL of PCR amplification product with 8.5 μL of formamide and 0.5 μL of molecular weight internal standard (T-500 Size Standard); denature the mixture at 95°C for 3 minutes, then cool it on ice for 3 minutes; use the aforementioned genetic analyzer to perform genotyping detection on 19 mRNA / cInDel markers. Obtain the electrophoretic migration parameters of different alleles at each locus using capillary electrophoresis, and based on this, write the corresponding Bin and Panel files according to the format requirements of GeneMapperID v3.2.1 software to create the electrophoretic analysis method.

[0058] The typing profiles of the five bodily fluid cDNA samples obtained using the above method are as follows: Figure 1 As shown.

[0059] Example 2

[0060] The cDNA samples from 10 semen, saliva, vaginal fluid, menstrual blood, and venous blood were tested using the method provided in Example 1.

[0061] Semen, saliva, vaginal fluid, 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 amplified using the method described in Example 1, employing a 19-mRNA / cInDel labeling technique. All samples yielded effective amplification products on their respective body fluid-specific labels and were successfully genotyped.

[0062] Example 3

[0063] This embodiment provides forensic verification of the method provided in Embodiment 1. The specific experiments and results are as follows:

[0064] In accordance with the requirements of the Scientific Working Group for DNA Analysis Methods (SWGDAM), the consistency, sensitivity, and species specificity of the method constructed in Example 1 were studied.

[0065] Forensic verification results showed that the method constructed in Example 1 exhibited high consistency in the detection results of five bodily fluids, with good tissue specificity; it also demonstrated high sensitivity, successfully detecting RNA samples of 2.5 ng or more. Figures 2-6 It exhibits strong species specificity, with no specific products detected in venous blood and oral swabs from nine common non-human samples (goose, donkey, chicken, sheep, pig, cow, dog, cat, and pigeon); it can detect mixed samples of semen and saliva, and semen and vaginal fluid, and has successfully genotyped them. Figures 7-8 ).

[0066] As can be seen from the above embodiments, the primer composition, method, and kit based on capillary electrophoresis technology for detecting body fluid-specific mRNA-cInDel markers provided by the present invention can be applied to determine the body fluid type of five body fluids and can provide InDel gene typing results of body fluid donors, providing a new detection method for forensic body fluid identification.

[0067] 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. A primer combination for a genetic marker used in body fluid identification, characterized in that, The genetic markers include mRNA markers and cInDel markers on mRNA; The primer combination includes primer pairs that amplify 19 genetic markers, as shown below: 。 2. The primer combination according to claim 1, characterized in that, In the primer pairs that amplify the 19 genetic markers, at least one primer in each primer pair is labeled with a fluorescent dye, which is selected from FAM, HEX, TAMRA and ROX.

3. The primer combination according to claim 2, characterized in that, The SLC4A1, ALAS2, STATH, KRT4, ODF1, and COL6A3 are labeled with FAM; the HTN3, LPAR3, CYP2B7P1, GPAT3, Lcris, and FAM83D are labeled with HEX; the PRM1, SEMG1, MMP11, and RPS4Y1 are labeled with TAMRA; and the TCP11, STC1, and XIST are labeled with ROX.

4. The use of a primer combination as described in any one of claims 1-3 in the preparation of a kit for identifying body fluids.

5. A reagent kit for identifying body fluids, characterized in that, Includes the primer combination described in any one of claims 1-3.

6. The reagent kit according to claim 5, characterized in that, The kit also includes 2×Multiplex PCRMaster Mix, 5×Q-Solution, and deionized water.

7. A method for identifying bodily fluids, characterized in that, Includes the following steps: RNA was extracted from the body fluids to be tested and reverse transcribed into cDNA; Using the cDNA as a template, PCR amplification was performed using the primer combination described in any one of claims 1-3 to obtain the amplification product; The amplification products are used for typing to determine the body fluid type of the tested fluid.

8. The identification method according to claim 7, characterized in that, The PCR amplification reaction system includes: 7.5 μL 2×Multiplex PCR Master Mix, 1.5 μL 5×Q-Solution, 3 μL deionized water, 1.5 μL primer mixture, and 1.5 μL cDNA.

9. The identification method according to claim 7, characterized in that, The PCR amplification reaction program includes: 95℃ pre-denaturation for 15 minutes; 94℃ denaturation for 30 seconds, 60℃ annealing for 1 minute and 30 seconds, 72℃ extension for 1 minute and 30 seconds, 35 cycles; and 60℃ final extension for 60 minutes.

10. The use of a primer combination as described in any one of claims 1-3 or a kit as described in any one of claims 5-6 in the identification of body fluids.

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