RPA-CRISPR-Cas12a-LFD body fluid traceability detection method based on test strip

Abstract

The invention discloses a detection method for forensic body fluid traceability. According to the method, peripheral blood, menstrual blood, vaginal secretion, semen and saliva are detected by using an RPA-CRISPR-Cas12a-LFD detection system and utilizing mRNA markers of HBB, MMP10, DKK4, AKAP4 and HTN3 genes. The detection method can realize accurate traceability of the sample and effective detection of the mixed body fluid, is short in detection time, can complete the test only in 40 minutes, is high in sensitivity, simple and convenient to operate and high in body fluid traceability efficiency, gets rid of dependence on large-scale equipment, and is beneficial to popularization and application in primary forensic DNA laboratories and biological research laboratories.

Description

Technical Field

[0001] This invention relates to the field of forensic technology, and in particular to a test strip-based RPA-CRISPR-Cas12a-LFD body fluid traceability detection method. Background Technology

[0002] Bodily fluid (stain) samples left at crime scenes can provide valuable information for case investigations. Identifying the type of bodily fluid (stain), i.e., bodily fluid tracing, can determine the nature of the case and provide clues for scene reconstruction and case deduction. For example, testing a sample for semen or vaginal secretions suggests a possible sexual assault case. With the development of molecular biology, many new methods for bodily fluid tracing have emerged, such as DNA methylation pattern analysis, transcriptome expression level analysis, and microbiome detection. Among these methods, messenger RNA (mRNA) molecular marker expression pattern analysis is considered a promising method for bodily fluid tracing due to its high sensitivity and specificity.

[0003] Currently, transcriptomics detection techniques mainly include reverse transcription-polymerase chain reaction (RT-qPCR), capillary electrophoresis (CE), and high-throughput sequencing. These methods can amplify several mRNA markers in the same reaction, achieving the goal of simultaneously detecting the expression levels of multiple mRNA markers for body fluid tracing. Furthermore, high-throughput sequencing allows for primer design unaffected by amplicon overlap, enabling the detection of more RNA molecules at once and obtaining RNA sequence information. However, these methods all have certain limitations. RT-qPCR requires a stable internal reference gene and its data interpretation is not intuitive; capillary electrophoresis has a long experimental procedure, failing to meet the requirements for rapid detection; high-throughput sequencing has high sample quality requirements and is costly. Moreover, these methods rely on large-scale instruments and equipment, making the reactions inconvenient. Therefore, a new technology is needed to meet the requirements of accurate, rapid, and convenient body fluid tracing.

[0004] Recombinase polymerase amplification (RPA) is a technique that can amplify nucleic acids at a constant temperature. The CRISPR / Cas system is an adaptive immune system found in prokaryotes, composed of CRISPR (clustered regularly spaced short palindromic repeats) and Cas (CRISPR-associated proteins). Among them, Cas12a protein is currently the most widely used Cas protein in nucleic acid detection. With the help of crRNA, Cas12a can recognize double-stranded DNA sequences with TTTN structures, thereby initiating the cleavage of single-stranded DNA modified with fluorescent groups, and detecting nucleic acids based on changes in fluorescence signals.

[0005] The purpose of this invention is to combine RPA, CRISPR-Cas12a and lateral flow immunochromatography (LFD) to construct a body fluid tracing and detection system, bridging the gap between laboratory accuracy and field applicability, and providing an accurate detection solution for tracing common body fluids in forensic medicine, as well as for difficult and complex samples such as degradation, putrefaction and mixing that are common in judicial practice. Summary of the Invention

[0006] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the objective of the present invention is to provide a test strip-based RPA-CRISPR-Cas12a-LFD body fluid tracing detection method, providing a high-resolution forensic body fluid tracing detection scheme for accurately identifying the sources of common forensic body fluids (peripheral blood, menstrual blood, vaginal secretions, semen, and saliva) and mixed body fluids.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A first aspect of the present invention provides a method for identifying bodily fluids, the method comprising the following steps:

[0009] (1) Obtain the nucleic acid from the sample to be tested;

[0010] (2) Use the nucleic acid from step (1) to perform recombinase polymerase amplification to obtain the amplification product;

[0011] (3) The amplification product described in step (2) is subjected to a CRISPR-Cas reaction to obtain the reaction product;

[0012] (4) Detect the reaction product described in step (3) and identify the body fluid based on the detection results;

[0013] The recombinase polymerase amplification system includes primer pairs with sequences as shown in SEQ ID NO:1-2, SEQ ID NO:3-4, SEQ ID NO:5-6, SEQ ID NO:7-8 and SEQ ID NO:9-10;

[0014] The primer pair targets and amplifies the HBB, MMP10, DKK4, AKAP4, and HTN3 genes in the body fluid.

[0015] In some embodiments of the present invention, the primer pairs with sequences such as SEQ ID NO:1-2 amplify the HBB gene in body fluids.

[0016] In some embodiments of the present invention, the primer pairs with sequences such as SEQ ID NO:3-4 amplify the MMP10 gene in body fluids.

[0017] In some embodiments of the present invention, the primer pair with the sequence shown in SEQ ID NO:5-6 amplifies the DKK4 gene in body fluid.

[0018] In some embodiments of the present invention, the primer pair with the sequence shown in SEQ ID NO:7-8 amplifies the AKAP4 gene in body fluid.

[0019] In some embodiments of the present invention, the primer pairs with sequences as shown in SEQ ID NO:9-10 amplify the HTN3 gene in body fluids.

[0020] In some embodiments of the present invention, the HBB gene is used to identify peripheral blood samples.

[0021] In some embodiments of the present invention, the MMP10 gene is used to identify menstrual blood samples.

[0022] In some embodiments of the present invention, the DKK4 gene is used to identify vaginal secretion samples.

[0023] In some embodiments of the present invention, the AKAP4 gene is used to identify semen samples.

[0024] In some embodiments of the present invention, the HTN3 gene is used to identify saliva samples.

[0025] In some embodiments of the present invention, the detection method includes fluorescence signal detection or immunochromatographic detection.

[0026] In some embodiments of the present invention, the fluorescence signal detection identifies body fluids based on changes in fluorescence intensity.

[0027] In some embodiments of the present invention, the immunochromatography includes lateral flow immunochromatography or longitudinal flow immunochromatography.

[0028] In some embodiments of the present invention, the immunochromatographic assay includes the use of a lateral flow immunochromatographic apparatus.

[0029] In some embodiments of the present invention, the use of the lateral flow immunochromatographic apparatus includes mixing the CRISPR-Cas reaction product and the chromatography solvent, and then performing detection using the lateral flow immunochromatographic apparatus.

[0030] In some embodiments of the present invention, the chromatography solvent includes at least one of PBS, sucrose solution, polyvinylpyrrolidone, and Tween.

[0031] In some embodiments of the present invention, the preparation of the lateral flow immunochromatographic apparatus includes the following steps:

[0032] (1) The latex microspheres were conjugated with rabbit anti-FITC antibody and sprayed onto the conjugate pad;

[0033] (2) Recombinant streptavidin (102D) and recombinant streptavidin (102T), and purified goat anti-rabbit IgG antibody were sprayed onto nitrocellulose membranes and used as C-line and T-line, respectively.

[0034] (3) The binding pad of (1) and the nitrocellulose membrane of (2) and the absorbent pad are pasted onto a PVC base plate to prepare a lateral flow immunochromatographic device.

[0035] In some embodiments of the present invention, the mass ratio of recombinant streptavidin (102D) to recombinant streptavidin (102T) used is 6-10:3-10.

[0036] In some embodiments of the present invention, the mass ratio of recombinant streptavidin (102D) to recombinant streptavidin (102T) used is 6-8:3-10.

[0037] In some embodiments of the present invention, the concentration of the recombinant streptavidin (102D) used is 1.5-4.5 mg / mL.

[0038] In some embodiments of the present invention, the concentration of the recombinant streptavidin (102T) used is 2-5 mg / mL.

[0039] In some embodiments of the present invention, when the lateral flow immunochromatographic device detects the presence of the C line, body fluid identification is performed based on the presence or absence of the T line.

[0040] In some embodiments of the present invention, the types of bodily fluids include peripheral blood, menstrual blood, vaginal secretions, semen, and saliva.

[0041] In some embodiments of the present invention, the CRISPR-Cas reaction system comprises crRNA with sequences as shown in SEQ ID NO:11-15.

[0042] In some embodiments of the present invention, the CRISPR-Cas reaction system comprises a DNA probe.

[0043] In some embodiments of the present invention, the DNA probe includes a probe with a biotin or BHQ1 label at the 3' end and a FITC or FAM label at the 5' end.

[0044] In some embodiments of the present invention, the sequence of the DNA probe is FTIC-TTATTT-biotin.

[0045] In some embodiments of the present invention, the sequence of the DNA probe is FAM-TTATTT-BHQ1.

[0046] In some embodiments of the present invention, the reaction conditions for the recombinase polymerase amplification are 37°C-45°C for 20-45 minutes.

[0047] In some embodiments of the present invention, the CRISPR-Cas reaction conditions are 37°C-45°C for 20-45 minutes.

[0048] In some embodiments of the present invention, the reaction system for recombinase polymerase amplification includes: 20-30 μL of A buffer, 2-4 μL of 10 μM forward and reverse primers, 1-5 μL of nucleic acid to be tested, 2-5 μL of B buffer, and ddH2O added to 50 μL.

[0049] In some embodiments of the present invention, the system of the CRISPR-Cas reaction is shown in the table below.

[0050]

[0051] Add DEPC water to a total of 30 μL.

[0052] A second aspect of the present invention provides a reagent for identifying bodily fluids, the reagent comprising at least one of primers, a DNA probe, and crRNA; the primers comprising primer pairs with sequences as shown in SEQ ID NO:1-2, SEQ ID NO:3-4, SEQ ID NO:5-6, SEQ ID NO:7-8, and SEQ ID NO:9-10.

[0053] In some embodiments of the present invention, the crRNA includes crRNA with sequences as shown in SEQ ID NO:11-15.

[0054] A third aspect of the invention provides a kit comprising the above-described reagents, the kit further comprising at least one of Cas protein and polymerase.

[0055] In some embodiments of the present invention, the Cas protein includes LbCas12a, AsCas12a, or BbCas12a.

[0056] A fourth aspect of the present invention provides the application of the above-described reagents or kits in the identification of body fluids, wherein the types of body fluids include peripheral blood, menstrual blood, vaginal secretions, semen, and saliva.

[0057] The beneficial effects of this invention are:

[0058] 1. The five specific mRNA markers of this invention can be used for forensic body fluid tracing with high accuracy and sensitivity.

[0059] 2. The RPA-CRISPR-Cas12a-LFD detection system constructed in this invention can accurately trace the source of five types of bodily fluids: peripheral blood, menstrual blood, vaginal secretions, semen, and saliva. It can effectively detect mixed bodily fluids. The sensitivity of HBB and MMP10 labeling reaches 0.1ng, and the sensitivity of AKAP4, DDK4, and HTN3 labeling is 1ng. It has a strong detection capability for trace samples.

[0060] 3. The detection method constructed in this invention is simple to operate, has a short detection time (detection can be completed in 40 minutes), high efficiency in tracing body fluids, and eliminates the dependence on large-scale equipment, which is conducive to its promotion and application in grassroots forensic DNA laboratories and biological research laboratories. Attached Figure Description

[0061] Figure 1 Fluorescence signal curves for RPA reaction time optimization experiments.

[0062] Figure 2 Fluorescence signal curves for RPA reaction temperature optimization experiments.

[0063] Figure 3 The fluorescence signal curves of the CRISPR-Cas12 reaction over 40 minutes are shown for different samples.

[0064] Figure 4 Fluorescence signal curves for the CRISPR-Cas12 reaction temperature optimization experiment.

[0065] Figure 5 Experimental results for optimizing the LFD reaction in the immunochromatographic lateral flow apparatus. A shows the results of tests with different concentrations of recombinant streptavidin (102D) and different amounts of DNA probe; B shows the results of positive standards and no DNA probe (negative control) at a recombinant streptavidin concentration of 3.0 mg / mL; C shows the results of tests with different concentrations of recombinant streptavidin (102T); D shows the results of tests using recombinant streptavidin (102D) and recombinant streptavidin (102T) in combination at different concentrations; E shows the results of negative controls and positive standards using 3.5 mg / mL recombinant streptavidin (102D) and 5.0 mg / mL recombinant streptavidin (102T) in combination.

[0066] Figure 6 Results of fluid traceability performance of the optimized RPA-CRISPR-Cas12a-LFD detection system using HBB and MMP10 markers. PB: peripheral blood; MB: menstrual blood; VS: vaginal secretions; SE: semen; SA: saliva.

[0067] Figure 7 Results of fluid tracing efficacy of the optimized RPA-CRISPR-Cas12a-LFD detection system using DKK4 and AKAP4 markers. PB: peripheral blood; MB: menstrual blood; VS: vaginal secretions; SE: semen; SA: saliva.

[0068] Figure 8 Results of fluid traceability performance of the optimized RPA-CRISPR-Cas12a-LFD detection system using HTN3 labeling. PB: peripheral blood; MB: menstrual blood; VS: vaginal secretions; SE: semen; SA: saliva.

[0069] Figure 9 The sensitivity results for the optimized RPA-CRISPR-Cas12a-LFD detection system are shown. The number under each test strip represents the amount of RNA used, in ng. PBS: Peripheral blood - sensitivity sample; MBS: Menstrual blood - sensitivity sample; VSS: Vaginal secretions - sensitivity sample; SES: Semen - sensitivity sample; SAS: Saliva - sensitivity sample.

[0070] Figure 10 To test the results of mixed samples using the optimized RPA-CRISPR-Cas12a-LFD detection system. PB: peripheral blood; MB: menstrual blood; VS: vaginal secretions; SE: semen; SA: saliva. Detailed Implementation

[0071] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0072] Example 1: Optimization of RPA and CRISPR-Cas12a reaction conditions

[0073] In this embodiment, peripheral blood, menstrual blood, vaginal secretions, semen, and saliva samples were amplified and detected using RPA and CRISPR-Cas12a technologies. The mRNA markers used were HBB (peripheral blood), MMP10 (menstrual blood), DKK4 (vaginal secretions), AKAP4 (semen), and HTN3 (saliva), respectively. The sequences of primers, probes, and crRNA used in the RPA-CRISPR-Cas12a experiment are shown in Table 1 below.

[0074] Table 1. Sequences of primers, probes, and crRNA used in RPA-CRISPR-Cas12a detection.

[0075]

[0076] The specific experimental steps in this embodiment are as follows.

[0077] (1) Collect three samples each of peripheral blood, menstrual blood, vaginal secretions, semen and saliva. Use the RNAiso Plus kit (TaKaRa, Tokyo, Japan) to extract total RNA from 200 μL of saliva, blood, semen, menstrual blood samples and oral and vaginal swabs.

[0078] (2) Perform the RPA reaction using the RNA isothermal rapid amplification kit (AMP Future): Add 29.4 μL of Abuffer, 2 μL of forward and reverse primers with a primer concentration of 10 μM, 1-5 μL of RNA, and 9.1-13.1 μL of ddH2O to the dry powder reaction tube until the total reaction volume is 47.5 μL; finally, add 2.5 μL of B buffer, mix thoroughly, centrifuge, and incubate in a constant temperature water bath at 42℃ for 20 minutes.

[0079] (3) CRISPR reaction using LbCas12a protein (Aidi gene): Prepare the reaction system in a 0.2 mL enzyme-free EP tube according to the CRISPR-Cas12a reaction system shown in Table 2 below. Mix the prepared reaction system, centrifuge, and perform the reaction on a real-time quantitative PCR instrument at 42℃ for 20 minutes. The fluorescence channel is the FAM channel, and fluorescence is collected every 30 seconds.

[0080] Table 2 CRISPR-Cas12a Reaction Ratio Table

[0081]

[0082]

[0083] The initial reaction conditions were set as follows: RPA was reacted at 42°C for 30 minutes, and CRISPR-Cas12a was reacted at 37°C for 30 minutes. Individual reaction conditions were optimized sequentially in the order of RPA reaction time, RPA reaction temperature, CRISPR-Cas12a reaction time, and CRISPR-Cas12a reaction temperature. The RPA reaction time gradients were 20, 25, 30, 35, and 40 minutes; the RPA reaction temperature gradients were 25, 30, 37, and 42°C; the CRISPR-Cas12a reaction time gradients were 20, 25, 30, 35, and 40 minutes; and the CRISPR-Cas12a reaction temperature gradients were 25, 30, 37, and 42°C. Three samples were used for each type of body fluid, and each sample was tested three times at each temperature gradient.

[0084] Experimental results are as follows Figure 1-4 As shown, in the optimization of RPA reaction time and temperature, the fluorescence signal values ​​of most samples were within 20 minutes ( Figure 1 ), 42℃ Figure 2 The reaction reached a plateau. Therefore, 20 minutes and 42°C were ultimately chosen as the reaction conditions for RPA. In the optimization of CRISPR-Cas12a reaction time and temperature, 20 minutes was selected. Figure 3 ), 42℃ Figure 4 () as the reaction conditions for CRISPR-Cas12a.

[0085] Example 2: Optimization of the Lateral Flow Immunochromatographic Device (LFD)

[0086] This embodiment provides an optimization of the immunochromatographic sideflow device LFD, and the specific experimental steps are as follows.

[0087] 1. Assembly of LFD

[0088] First, the latex microspheres were conjugated with the rabbit anti-FITC antibody, and the specific steps are as follows:

[0089] Add 500 μL of MES solution and 10 μL of latex microspheres (Shanghai Xiongtu Biotechnology Co., Ltd.) to a 1.5 mL centrifuge tube and sonicate for 1-2 minutes. Then add 15 μL of 5 mg / mL EDC solution and 15 μL of 5 mg / mL NHS solution. Sonicate for 1-2 minutes and vortex for 15 minutes. After vortexing, centrifuge at 4°C and 14000 rpm for 15 minutes. After centrifugation, collect the supernatant and add 500 μL of labeling buffer (Shanghai Xiongtu Biotechnology Co., Ltd.), activated latex microspheres (Shanghai Xiongtu Biotechnology Co., Ltd.), and 2 μL of rabbit anti-FITC antibody (Beijing Biosen Biotechnology Co., Ltd.). Vortex for 1 hour, then add 10 μL of 20% bovine serum albumin solution and vortex for 3 hours. After vortexing, centrifuge at 4°C and 14000 rpm for 15 minutes. Finally, remove the supernatant, add 20 μL of stock solution (containing 0.05 mol / L Tris-HCl solution, 1% BSA, 5% sucrose, 0.5% Tween-20, 1% Procline 300, 0.1% sodium azide, pH 8.0), and store at 4°C.

[0090] After conjugating latex microspheres with rabbit anti-FITC antibody, LFD was assembled, and the specific steps are as follows:

[0091] The LFD consists of a sample pad, a conjugation pad, a nitrocellulose (NC) membrane, an absorbent pad, and a PVC substrate. First, the prepared latex microspheres conjugated with rabbit anti-FITC antibody are sprayed onto the conjugation pad. Recombinant streptavidin (102D) (Beijing Bosi Technology Co., Ltd.), recombinant streptavidin (102T) (Beijing Bosi Technology Co., Ltd.), and purified goat anti-rabbit IgG antibody (Beijing Bio-Sens Biotechnology Co., Ltd.) are diluted with PBS solution (10 mmol / L, pH 7.4) to 3.5 mg / mL, 5.0 mg / mL, and 1 mg / mL, respectively. The diluted recombinant streptavidin (102D) and recombinant streptavidin (102T) are mixed at a volume ratio of 7:3 as the C-line component of the LFD, and the purified goat anti-rabbit IgG antibody is the T-line component. Recombinant streptavidin and purified goat anti-rabbit IgG antibody are sprayed onto the NC membrane at a spraying rate of 4 μL / cm, respectively, as the C-line and T-line of the LFD. Place the NC film in an oven and dry it overnight at 45°C. Remove the dried NC film from the oven and attach the NC film, bonding pad, and absorbent pad to the PVC base plate. Cut the attached PVC base plate into 3.95mm wide strips using a programmable adhesive tape and insert them into custom plastic cards to create the LFD (Liquid Forming Device). Place the LFD in an aluminum foil bag and store at 4°C.

[0092] 2. LFD Optimization

[0093] Since the relationship between the ratio of latex microspheres to rabbit anti-FITC antibody, the concentration of recombinant streptavidin, and the concentration of the DNA probe used in the CRISPR reaction can affect whether LFD produces false positive results, the concentrations of recombinant streptavidin and the DNA probe were optimized. In the experiments involving RPA and CRISPR-Cas12a binding to LFD, the DNA probe sequence used in the CRISPR reaction was FITC-TTAT TT-biotin (probe2).

[0094] The volume ratio between the fixed latex microspheres and rabbit anti-FITC antibody was set to 10:2, and then the amount of DNA probe (probe2) and the concentration of recombinant streptavidin were adjusted. DNA probe dosage gradients of 6 μL, 8 μL, 10 μL, 12 μL, 14 μL, and 16 μL were set, with a concentration maintained at 2 μM. Simultaneously, recombinant streptavidin (102D) concentration gradients of 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, and 3.0 mg / mL were set for testing; recombinant streptavidin (102T) concentration gradients of 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, and 5.0 mg / mL were set for testing. Recombinant streptavidin (102D) and recombinant streptavidin (102T) were fixed at concentrations of 1.5 mg / mL + 5.0 mg / mL, 2.0 mg / mL + 5.0 mg / mL, 2.5 mg / mL + 5.0 mg / mL, 3.0 mg / mL + 5.0 mg / mL, and 3.5 mg / mL + 5.0 mg / mL, respectively, for testing. In the optimized reaction under the above LFD conditions, the RPA product was replaced with 5 μL of DEPC water as a negative control, and a probe with the FITC-modified sequence FIT C-TTATTT was used as a positive standard.

[0095] The specific steps for LFD are as follows: Following the experimental design described above, add 70 μL of chromatography buffer (PBS + 1% sucrose + 1% polyvinylpyrrolidone (PVP) + 1% Tween) to the DNA probe probe2 (used as the sample). Then, drop the buffer onto the sample pad of the test strip and observe the results after 3-5 minutes. A very weak T line indicates a negative result, while a distinct T line indicates a positive result.

[0096] Experimental results are as follows Figure 5 As shown, Figure 5 As shown in Figure A, no false positive results were observed when the concentration of recombinant streptavidin (102D) was 3.0 mg / mL. At the same time, the amount of DNA probe (probe2) used did not affect the detection results of C and T lines. Therefore, the amount of DNA probe used was set to 6 μL.

[0097] and Figure 5 The positive standard sample B showed a false negative result. When the CRISPR-Cas12a reaction product (DEPC water, negative control) without DNA probe was used in the experiment, the results showed that LFD only showed a C line (theoretically, only a T line should appear). Figure 5 As shown in Figure C, false positive results were observed when using recombinant streptavidin (102T), which has a slightly weaker binding affinity, alone. Figure 5As shown in Figure D, false positive results still occurred when recombinant streptavidin (102D) and recombinant streptavidin (102T) were administered at concentrations of 1.5 mg / mL + 5.0 mg / mL, 2.0 mg / mL + 5.0 mg / mL, 2.5 mg / mL + 5.0 mg / mL, and 3.0 mg / mL + 5.0 mg / mL, respectively. However, the result was normal and no false positives were observed at 3.5 mg / mL + 5.0 mg / mL. Figure 5 As shown in Figure E, when the volume ratio of recombinant streptavidin (102D, 3.5 mg / mL) to recombinant streptavidin (102T, 5.0 mg / mL) was 7:3, both the negative control and the positive standard group produced correct results.

[0098] In summary, the final reaction conditions for RPA and CRISPR-Cas12a were 20 minutes, 42°C, and 6 μL of DNA probe (probe2). The reaction conditions for LFD were: recombinant streptavidin (102D, 3.5 mg / mL) and recombinant streptavidin (102T, 5.0 mg / mL) were mixed at a volume ratio of 7:3.

[0099] Example 3: Performance Evaluation of the RPA-CRISPR-Cas12a-LFD Detection System for Body Fluid Traceability

[0100] This embodiment evaluates the efficacy of the RPA-CRISPR-Cas12a-LFD detection system for tracing bodily fluids. Thirteen peripheral blood samples, thirteen menstrual blood samples, fifteen vaginal secretion samples, ten semen samples, and fifteen saliva samples were collected in this embodiment. Sample information is shown in Table 3 below. The optimized RPA-CRISPR-Cas12a and LFD detection systems from Examples 1 and 2 were used to evaluate the detection of different markers. The DNA probe used in the CRISPR-Cas12a reaction was the DNA probe probe2 from Example 4.

[0101] Table 3 Information on samples used for specificity verification

[0102]

[0103] The LFD results for all samples with five mRNA markers are as follows: Figures 6 to 8 As shown. Figure 6 It can be seen that only HBB markers were detected in all 13 peripheral blood samples, while HBB and MMP10 markers were detected in all 13 menstrual blood samples. Figure 7 It can be seen that in 15 vaginal secretion samples, only DKK4 markers were detected in all samples. However, in 10 SE samples, AKAP4 markers were detected in all but two SE samples. Figure 7 The saliva-labeled HTN3 was detected in only 15 saliva-labeled samples. Figure 8 Experimental results show that the RPA-CRISPR-Cas12a-LFD detection system established in this application has high efficacy in forensic body fluid tracing.

[0104] Example 4: Sensitivity Evaluation of the RPA-CRISPR-Cas12a-LFD Detection System

[0105] This embodiment evaluates the sensitivity of the optimized RPA-CRISPR-Cas12a-LFD detection system from Examples 1 and 2. The DNA probe used in the CRISPR-Cas12a reaction is probe2 from Example 4. Peripheral blood, menstrual blood, vaginal secretions, semen, and saliva samples were collected, with three samples for each type of bodily fluid. Each gradient for each sample was tested three times for repeatability. Different amounts of RNA template (10 ng, 1 ng, 0.1 ng, 0.01 ng) were added to the RPA reaction according to the set RNA template amount. For each mRNA marker, sensitivity was considered only achieved if the corresponding mRNA marker was detected in ≥2 replicates across all three samples.

[0106] Experimental results are as follows Figure 9 As shown, in peripheral blood samples, PBS1 and PBS2 samples were detectable with an RNA input of 0.01 ng, while the limit of detection for PBS3 was 0.1 ng. Therefore, the sensitivity of HBB labeling in peripheral blood samples is 0.1 ng. In menstrual blood samples, the limit of detection for MMP10 labeling is 0.1 ng. In vaginal secretion samples, since VSS2 was undetectable at 0.1 ng, the sensitivity of DKK4 labeling in vaginal secretions is 1 ng. In semen and saliva samples, the minimum RNA input to detect all three samples was 1 ng, therefore the sensitivity of AKAP4 and HTN3 labeling is 1 ng.

[0107] Example 5: Detection of mixed samples using the RPA-CRISPR-Cas12a-LFD detection system.

[0108] This embodiment provides an optimized RPA-CRISPR-Cas12a-LFD detection system for detecting mixed samples. The DNA probe used in the CRISPR-Cas12a reaction is the DNA probe probe2 from Example 4. The five mixed sample types are: a mixture of peripheral blood and saliva, a mixture of peripheral blood and semen, a mixture of menstrual blood and saliva, a mixture of vaginal secretions and saliva, and a mixture of menstrual blood, vaginal secretions, and semen. Each type of mixed sample consists of three samples. Information on the mixed samples is shown in Tables 4-6 below.

[0109] Table 4 Information on mixed samples of peripheral blood and saliva, and peripheral blood and semen.

[0110]

[0111] Note: PB: peripheral blood; SE: semen; SA: saliva

[0112] Table 5 Information on mixed samples of menstrual blood and saliva, and vaginal secretions and saliva.

[0113]

[0114] Note: MB: menstrual blood; VS: vaginal discharge; SA: saliva.

[0115] Table 6 Information from a mixed sample of menstrual blood, vaginal discharge, and semen.

[0116]

[0117] Note: MB: menstrual blood; VS: vaginal discharge; SE: semen.

[0118] Figure 10 As can be seen, in the detection of mixed samples, peripheral blood marker HBB and saliva marker HTN3 were detected in all target mixed samples, while semen marker AKAP4 was not detected in any mixed samples. Menstrual blood marker MMP10 and vaginal secretion marker DKK4 were only detected in some target mixed samples. Specifically, MMP10 was detected in all mixed samples of menstrual blood and saliva, and in two triple mixed samples of menstrual blood + vaginal secretion + semen (MB + VS + SE1 and MB + VS + SE3). DKK4 was detected only in three mixed samples: VS + SA1, VS + SA3, and MB + VS + SE1.

[0119] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for identifying bodily fluids, characterized in that, The method includes the following steps: (1) Obtain the nucleic acid from the sample to be tested; (2) Use the nucleic acid from step (1) to perform recombinase polymerase amplification to obtain the amplification product; (3) The amplification product described in step (2) is subjected to a CRISPR-Cas reaction to obtain the reaction product; (4) Detect the reaction product described in step (3) and identify the body fluid based on the detection results; The recombinase polymerase amplification system includes primer pairs with sequences as shown in SEQ ID NO:1-2, SEQ ID NO:3-4, SEQ ID NO:5-6, SEQ ID NO:7-8 and SEQ ID NO:9-10; The primer pair targets and amplifies the HBB, MMP10, DKK4, AKAP4, and HTN3 genes in bodily fluids; the HBB gene is used to identify peripheral blood samples, the MMP10 gene is used to identify menstrual blood samples, the DKK4 gene is used to identify vaginal secretion samples, the AKAP4 gene is used to identify semen samples, and the HTN3 gene is used to identify saliva samples.

2. The method according to claim 1, characterized in that, The detection methods include fluorescence signal detection or immunochromatographic detection.

3. The method according to claim 2, characterized in that, The fluorescence signal detection identifies body fluids based on changes in fluorescence intensity.

4. The method according to claim 2, characterized in that, The immunochromatographic assay includes the use of a lateral flow immunochromatographic apparatus.

5. The method according to claim 4, characterized in that, When the lateral flow immunochromatographic device detects the presence of the C line, body fluid identification is performed based on the presence or absence of the T line.

6. The method according to claim 1, characterized in that, The types of bodily fluids include peripheral blood, menstrual blood, vaginal secretions, semen, and saliva.

7. The method according to claim 1, characterized in that, The CRISPR-Cas reaction system includes crRNA with sequences as shown in SEQ ID NO:11-15.

8. A reagent for identifying body fluids, characterized in that, The reagent includes at least one of primers, DNA probes, and crRNA; the primers include primer pairs with sequences as shown in SEQ ID NO:1-2, SEQ ID NO:3-4, SEQ ID NO:5-6, SEQ ID NO:7-8, and SEQ ID NO:9-10; preferably, the crRNA includes crRNA with sequences as shown in SEQ ID NO:11-15.

9. A kit comprising the reagent of claim 8, characterized in that, The kit also contains at least one of Cas protein and polymerase.

10. The application of the reagent of claim 8 or the kit of claim 9 in the identification of body fluids, characterized in that, The types of bodily fluids include peripheral blood, menstrual blood, vaginal secretions, semen, and saliva.

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