Primer combination for body fluid detection based on RT-LAMP technology, composite system and application

Abstract

The invention discloses a primer combination for body fluid detection based on an RT-LAMP technology, a composite system and application, and belongs to the technical field of forensic medicine identification. The kit comprises a housekeeping gene, a specific mRNA (messenger ribonucleic acid) site and a primer combination for amplifying a corresponding site, each group of primers in the primer combination comprises two inner primers, two outer primers and two loop primers, and the specific sequences of the primers are as shown in SEQ ID NO.1-60. The LAMP primer is designed aiming at the body fluid specific mRNA site, the expression level of mRNA is detected, and then a body fluid sample is recognized. The method is simple to operate, high in specificity, strong in tolerance, strong in recognition capability and short in time consumption, can obtain a body fluid type result within 30 minutes, and can be applied to the field of forensic body fluid identification and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of forensic identification, and particularly relates to a primer combination for body fluid detection based on RT-LAMP technology, a composite system and a use. BACKGROUND

[0002] Since STR (short tandem repeat) was discovered, it has become the gold standard for personal identification in the field of forensic science. However, STR analysis can only provide information about the identity of a suspect and cannot determine the source of biological evidence. This suspect identification that ignores the source of the organization may lead to providing false clues in a case. In forensic cases, blood, semen, saliva, vaginal secretions, and menstrual blood are common physical evidence. These body fluid samples can not only be used for individual identification, but also help determine the nature of the case, indicate the direction of the case, and reconstruct the crime scene. Therefore, body fluid identification is of great significance in forensic practice. With the development of molecular biology technology, new body fluid-specific molecular biomarkers have been developed and screened.

[0003] mRNA is the earliest molecular genetic marker used for body fluid identification. During growth and development, cells differentiate into different functional terminally differentiated cells. In this process, different cells express different proteins, so the expression pattern of different functional cells should be unique, which can be verified by detecting the presence and relative abundance of specific mRNA. According to the expression profile of different tissue cells, mRNA expression markers specific to tissue cells can be screened. These mRNA markers have body fluid specificity. In the 1990s, forensic scientists began to explore body fluid-specific mRNA markers. In recent decades, forensic scientists have screened a large number of mRNA markers that are stably expressed in tissues and cells, and have analyzed and evaluated their specificity and sensitivity. At present, mRNA is the most in-depth and commonly used genetic marker in body fluid identification.

[0004] In resource-poor areas or when cases are urgent, on-site rapid body fluid identification can provide timely and effective information, saving time and cost. Compared with conventional PCR technology, isothermal amplification technology has the advantages of no need for strict temperature control and short amplification time, and can be combined with various visualization schemes (such as lateral flow test strips and fluorescence coloration) to observe the results, making it suitable for on-site rapid detection. Commonly used isothermal amplification techniques include RPA (Recombinase polymerase amplification), LAMP (Loop-mediated isothermal amplification), RCA (Rolling circle amplification), and NASBA (Nucleic acid sequence-based amplification). Kubo et al. developed an optimized reverse transcription RPA detection method for blood HBB mRNA markers, which can complete target site detection in a single tube, but still relies on laboratory equipment and only contains one site. Liu et al. developed an RT-RPA-LFD detection method for two mRNA markers in saliva and vaginal secretions combined with lateral flow test strips, but still requires two-step amplification to complete mRNA marker detection, and the system still only contains two sites. Tetsuya et al. developed an RT-LAMP rapid detection method for three mRNA markers in blood, semen, and saliva. Kimberly et al. developed an RT-LAMP detection method for four mRNA markers in blood, semen, saliva, and vaginal secretions and combined with smartphone software for colorimetric judgment of results. However, these methods have the following limitations: limited types of body fluids and target markers, and inability to completely eliminate dependence on laboratory equipment (some even require two-step amplification). These factors severely limit their application in forensic on-site rapid detection. Despite the increasing interest in rapid body fluid detection schemes, existing methods still have the problems of insufficient marker coverage and strong instrument dependence, and too few markers can significantly affect the accuracy of body fluid identification.

[0005] Although the patent application (CN202310361860.7, "A composite amplification system for identifying mixed body fluids, primers and kits thereof") discloses detection sites for four types of body fluids, in actual application, we found that the site combination of this patent has the following technical limitations: 1) The detection sensitivity for saliva and vaginal secretions is only 25 ng, while the sensitivity for blood and semen is 0.1 ng, and high-quality RNA templates are required, limiting the identification ability of low templates and degraded samples; 2) The kit still relies on laboratory professional equipment, with complex operation steps and long time-consuming, which cannot meet the needs of forensic on-site rapid detection, i.e., "sample in - result out". SUMMARY

[0006] In order to solve the above problems in the prior art, the application provides a primer combination, a composite system and a use for body fluid detection based on RT-LAMP technology.

[0007] To achieve the above object, the technical scheme adopted by the application to solve its technical problem is: The application aims to provide a composite system for body fluid detection based on RT-LAMP technology, which comprises a housekeeping gene, a specific mRNA site and a primer combination for amplifying the corresponding site. The specific mRNA site comprises HBB, HBA, MSMB, KLK3, CYP2B7P, SPINK5, KRT4 and KRT13; the housekeeping gene comprises GAPDH and B2M; each group of primers in the primer combination comprises two inner primers, two outer primers and two loop primers, and the specific sequences are shown in SEQ ID NO. 1-60.

[0008] Another object of the application is to provide a primer combination for body fluid detection based on RT-LAMP technology, wherein each group of primers in the primer combination comprises two inner primers, two outer primers and two loop primers, and the specific sequences are shown in SEQ ID NO. 1-60. HBB-F3: CTCACCTGGACAACCTCAAG (SEQ ID NO.1); HBB-B3: TTGTGGGCCAGGGCATTA (SEQ ID NO.2); HBB-FIP:CCAGGAGCCTGAAGTTCTCAGGGGCACCTTTGCCACACTG (SEQ ID NO.3); HBB-BIP:TGTGTGCTGGCCCATCACTTCCAGCCACCACTTTCTGAT (SEQ ID NO.4); HBB-LF: TGTCACAGTGCAGCTCACT (SEQ ID NO.5); HBB-LB: GCAAAGAATTCACCCCACCA (SEQ ID NO.6); HBA-F3: ATGGTGCTGTCTCCTG (SEQ ID NO.7); HBA-B3: AGCCAGGAACTTGTCC (SEQ ID NO.8); HBA-FIP:GGCTCAGGTCGAAGTGCAGGCCCTGGAGAGGA (SEQ ID NO.9); HBA-BIP: ACGTGGACGACATGCCAGTGGCTTAGGAGCTTGA (SEQ ID NO.10); HBA-LF: GAAGTAGGTCTTGGTGGTG (SEQ ID NO.11); HBB-LB: CACAAGCTTCGGGTGG (SEQ ID NO.12); MSMB-F3: GGAGTTCCAGGAGATTCA (SEQ ID NO.13); MSMB-B3: TTCTTCTCCACCACGATAT (SEQ ID NO.14); MSMB-FIP:CGTAGCAAGTGCATGTCTCAGCATGGATCTCAAAGGAAA (SEQ ID NO.15); MSMB-BIP:CTTGTTTCTACACCTGTGGGTTTCCTCCTTCTTGAAGATTCT(SEQ ID NO.16); MSMB-LF: GTTGTCAGTCTGCCACTC (SEQ ID NO.17); MSMB-LB: ATGACAAAGACAACTGCCA (SEQ ID NO.18); KLK3-F3: CATTGAACCAGAGGAGTTC (SEQ ID NO.19); KLK3-B3: TGATCCACTTCCGGTAAT (SEQ ID NO.20); KLK3-FIP:ACACAGCATGAACTTGGTCATTATTTCCAATGACGTGTGT (SEQ ID NO.21); KLK3-BIP:ACCTGCTCGGGTGATTCTCCATGACGTGATACCTTG (SEQ ID NO.22); KLK3-LF: TCTGAGGGTGAACTTGCG (SEQ ID NO.23); KLK3-LB: CACTTGTCTGTAATGGTGTGC (SEQ ID NO.24); CYP2B7P-F3: ACATCATCTGCTCCATCA (SEQ ID NO.25); CYP2B7P-B3: GTAGGTGTCGATGAGGTC (SEQ ID NO.26); CYP2B7P-FIP: AACAGCTGGCTGGATATAGTACCAAGATCAAGAGTTCCT (SEQ ID NO.27); CYP2B7P-BIP: GCTCTTCTCTGGCTTCTTGAATAAGCATTGATTTCCTGTAGG (SEQ ID NO.28); CYP2B7P-LF: TCTGGCAGAACAAGTTCAG (SEQ ID NO.29); CYP2B7P-LB: CACACAGGCAAGTTTACAAA (SEQ ID NO.30); SPINK5-F3: TATGAAGCTGTTTGTGGC (SEQ ID NO.31); SPINK5-B3: GAATTCTAGTTTCACCCTCTC (SEQ ID NO.32); SPINK5-FIP: GCATACATCCTGCTCTGGAGGTCCCAAATTGGTGTAAA (SEQ ID NO.33); SPINK5-BIP: TGGAAGACTTGGATGCACAAGCTTATTGCCATGCGTCTT (SEQ ID NO.34); SPINK5-LF: TGGATTACTGCTCTTACATTCC (SEQ ID NO.35); SPINK5-LB: AATGATCCTGTTCTTGGTCC (SEQ ID NO.36); KRT4-F3: GAGACCTACCTCAGTGTC (SEQ ID NO.37); KRT4-B3: CAACTCCACCTTGTTCAG (SEQ ID NO.38); KRT4-FIP: GGTCTTCAGCTCAGACTGCCTGAGGAAGCAGCTAGAT (SEQ ID NO. 39); KRT4-BIP: TGGAGGACTTCAAGACTAAGTATGCCACAAAGTCATTCTCGG (SEQ ID NO. 40); KRT4-LF: TTTGTCATTGCCCAAGGT (SEQ ID NO. 41); KRT4-LB: GATCAACAAACGCACAGC (SEQ ID NO. 42); KRT13-F3: GACTCATCAGCAGCATC (SEQ ID NO. 43); KRT13-B3: CTACGGACATCAGAAGTG (SEQ ID NO. 44); KRT13-FIP: GGTCTTCAGCTCAGACTGCCTGAGGAAGCAGCTAGAT (SEQ ID NO. 39); KRT13-BIP: TGGAGGACTTCAAGACTAAGTATGCCACAAAGTCATTCTCGG (SEQ ID NO. 40); KRT13-LF: TTTGTCATTGCCCAAGGT (SEQ ID NO. 41); KRT13-LB: GATCAACAAACGCACAGC (SEQ ID NO. 42); GAPDH-F3: GAGTCAACGGATTTGGTC (SEQ ID NO. 49); GAPDH-B3: CCACTTGATTTTGGAGGG (SEQ ID NO. 50); GAPDH-FIP: TTTGCCATGGGTGGAATCATATTTGTTGCCATCAATGACC (SEQ ID NO. 51); GAPDH-BIP: CCGTCAAGGCTGAGAACGCTCCTGGAAGATGGTGAT (SEQ ID NO. 52); GAPDH-LF: TGTAGTTGAGGTCAATGAAGG (SEQ ID NO. 53); GAPDH-LB: GCTTGTCATCAATGGAAATCC (SEQ ID NO.54); B2M-F3: CCATCCGACATTGAAGTTG (SEQ ID NO.55); B2M-B3: CCTCCATGATGCTGCTTA (SEQ ID NO.56); B2M-FIP: AGAAAGACCAGTCCTTGCTGACTGAAGAATGGAGAGAGAATTG (SEQ ID NO.57); B2M-BIP: TGAGTATGCCTGCCGTGTCATGTCTCGATCCCACTT (SEQ ID NO.58); B2M-LF: GACAAGTCTGAATGCTCCAC (SEQ ID NO.59); B2M-LB: TGTCACAGCCCAAGATAGT (SEQ ID NO.60).

[0009] Further, at least one primer in each primer set spans the exon junction region.

[0010] Further, the body fluid is at least one of blood, semen, saliva and vaginal secretion.

[0011] Another object of the present application is to provide a kit comprising the above primer combination.

[0012] Another object of the present application is to provide the use of the above primer combination, or the kit in the preparation of reagents for forensic identification.

[0013] Another object of the present application is to provide the use of the primer combination, or the kit in the preparation of preparations for body fluid detection and identification.

[0014] Another object of the present application is to provide a method for detecting body fluid based on RT-LAMP technology, comprising the following steps: (1) treating the sample to be tested with a lysis solution to obtain the RNA of the sample to be tested; (2) using the above primer combination, or the kit, using reverse transcription loop-mediated isothermal amplification technology to amplify the RNA of the sample to be tested, and determining whether the sample contains body fluid components according to the amplification results.

[0015] Furthermore, the amplification system includes components with the following concentrations: Tris-HCl 50mM / L, GLC 2.66%, dNTPs1.2~1.4mM / L, DT-40 0.83x, MgSO46mM / L, Tween-20 0.10%, HNB 0.06mM / L, F3 / B3 0.4μM / L, FIP / BIP 1.6~3.2μM / L, LF / LB 0.8~1.6μM / L, Bst3.0 0.32U / μL, and RI / RT 2U / μL.

[0016] Furthermore, the amplification reaction conditions were 65° C. and 30 min.

[0017] Furthermore, the lysate includes the following components at the following concentrations: Triton 0.5x, EVA 0.5x, KCL 75-100 mM, and KOH 20-25 mM.

[0018] Furthermore, the above reaction system, reaction procedure, and freeze-dried reagent loading are integrated into the integrated constant temperature nucleic acid detector mini7, which is equipped with a fluorescence recognition system, which can accurately identify the color change caused by the amplification reaction, and further determine the type of body fluid based on the positive reaction of the marker.

[0019] Beneficial effects of the present invention: This study designed LAMP primers targeting specific mRNA sites in body fluids to detect mRNA expression levels and identify body fluid samples. This method is simple to operate, highly specific, well tolerated, and capable of identifying body fluids in a short time. It can obtain body fluid typing results within 30 minutes and is applicable to forensic body fluid identification and other fields.

[0020] The RT-LAMP (Reverse Transcription-Based Loop-Mediated Isothermal Amplification) adopted in the application utilizes the combination of reverse transcriptase and DNA polymerase to detect RNA sequences, and the reaction involves 3 pairs of primers, including: internal primers, forward internal primer (FIP) and reverse internal primer (BIP); external primers, forward primer (F3) and reverse primer (B3); optional loop primers, forward loop primer (LF) and reverse loop primer (LB). The internal and external primers are combined with Bst DNA polymerase (which has high strand displacement activity at a temperature of 60-65℃) to form a dumbbell-shaped DNA structure. This structure can serve as a template for further amplification. After the addition of loop primers complementary to the dumbbell-shaped DNA, the amount of starting template in the LAMP reaction can be increased to 8 amplified DNA sequences. Therefore, the loop primers greatly improve the efficiency and sensitivity of the reaction and shorten the reaction time by 50%. The LAMP technology can complete the detection under the action of Bst DNA polymerase at a stable temperature of 60-65℃ for about 30 min. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Color change results of CYP2B7P marker detection of vaginal secretion samples; Figure 2 Sensitivity detection results of the RT-LAMP system constructed by the application; wherein, HBA and HBB are detection results in blood, MAMB and KLK3 are detection results in semen, KRT4 and KRT13 are detection results in saliva, CYP2B7P and SPINK5 are detection results in vaginal secretion; Figure 3 Specificity detection results of the RT-LAMP system constructed by the application on body fluids; Figure 4 Specificity detection results of the RT-LAMP system constructed by the application on RNA products; Figure 5 Real-time fluorescence signal reporting results of the mini7 integrated constant-temperature nucleic acid detector for vaginal secretion; Figure 6 Capillary electrophoresis results of the RNA body fluid identification kit for vaginal secretion samples. DETAILED DESCRIPTION

[0022] The specific embodiments of the present application are described below to facilitate the understanding of the present application for those skilled in the art, but it should be clear that the present application is not limited to the scope of the specific embodiments, and for those skilled in the art, it is obvious that various changes are within the spirit and scope of the present application defined and determined by the appended claims, and all the inventions utilizing the concept of the present application are within the scope of protection.

[0023] Example 1 Site screening, primer design 1. Site screening According to the literature reports, the body fluid specific mRNA sites HBB, HBA, MSMB, KLK3, CYP2B7P, SPINK5, KRT4, KRT13, and two housekeeping genes GAPDH and B2M are screened.

[0024] 2. Primer design According to the obtained sites, the corresponding RT-LAMP amplification primers are designed, each group of primers including two inner primers (FIP, BIP), two outer primers (F3, B3), and two loop primers (LF, LB); at least one primer in each group of primers spans the exon junction region, and the specific sequences of the primers are shown in SEQ ID NO. 1-60.

[0025] Example 2 RT-LAMP detection 1. Nucleic acid extraction Mix 1 μL of body fluid sample (blood, semen) or half of the swab (saliva, vaginal secretion) with 500 μL of 1x lysis solution (previously placed in the lysis chamber of the mini7 device), and incubate for 5 minutes. The composition of the lysis solution includes: 6.25 μL of 20% Triton, 6.25 μL of 20x EVA, 16 μL of 2500 mM / L KCL, 10 μL of 1000 mM / L KOH, and 461.5 μL of DEPC-H2O.

[0026] 2. Freeze-drying of reagents Quickly inject liquid nitrogen into the 2x LAMP reaction mixture to freeze the reagents into beads. Further freeze-drying is performed using a Pilot5-8 Pro freeze-dryer (Biocool, China). The mass percentage concentration of water in the freeze-dried reagent beads is 1.97% ± 0.15% as determined by a C30S Karl Ficher titrator (Mettler-Toledo, USA). Except for the lysis solution, all other required reagents are pre-freeze-dried.

[0027] 3. Loop-mediated isothermal amplification Press the mini7 sample box, press the lysed sample in the lysate chamber into 10 reaction chambers and mix with 2x amplification mix freeze-dried balls, add about 50 μL of lysed sample to each reaction chamber, detect one target in each reaction chamber, start the reaction by controlling the temperature of the heating module, and the reaction conditions are: 65°C, 30 min. The components of the 2x amplification mix freeze-dried ball include the following table, and each freeze-dried ball contains 11.2 μL of 2x amplification mix reagent.

[0028] Table 1 Amplification system

[0029] 4. Amplification signal recognition and analysis The Mini7 device is connected to a computer, and the gene-9660 software is used to receive the color change signal of the system caused by product amplification in real time, calculate the amplification signal curve, and observe the peak situation of the amplification curve to determine the positive and negative results.

[0030] Example 3 1. Sensitivity test The four kinds of body fluid RNA extracted by the organic method were diluted with a two-fold concentration difference to form a concentration gradient, and the sensitivity of the RT-LAMP system in different body fluids was determined using a qPCR instrument, and the results are shown in Figure 2 The reaction procedure is as follows: Table 2 Reaction procedure

[0031] As Figure 2 can be seen, the detection limit of the RT-LAMP body fluid identification system for blood is 25 pg, for semen is 4.5 ng, for vaginal secretion is 2.5 ng, and for saliva is 5 ng, with excellent sensitivity.

[0032] In addition, according to the results Figure 1 , the color of the CYP2B7P marker of the vaginal secretion sample changed before and after detection, proving that the reaction can be monitored by color change of the system.

[0033] 2. Specificity Different body fluid samples such as blood, semen, saliva, and vaginal secretion were collected, and RNA was extracted using the organic method. The body fluid specificity was verified using the RNA extract of different body fluid samples, and the specificity of the RNA primer set was verified using human genomic standard DNA (9947A), and the results are shown in Figure 3 and Figure 4 (all products are from mRNA template).

[0034] As shown in Figure 3 , the target product shows body fluid specificity. Cross-reaction is shown between saliva and vaginal secretion targets, and the results are consistent with Figure 6The electrophoresis results of the RNA body fluid identification kit were the same.

[0035] 3. Comparison of results with RNA body fluid identification kit The new RT-LAMP system was used to detect vaginal secretions. Figure 6 .

[0036] like Figure 6 As shown, the method of the present invention only requires 1 μL of body fluid and the results can be obtained in just 30 minutes, while the RNA body fluid identification kit requires 500 μL of body fluid or a whole swab to extract high-quality RNA, and it takes 6-7 hours to complete the detection of vaginal secretion samples.

[0037] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A composite system for body fluid detection based on RT-LAMP technology, characterized in that: It includes a housekeeping gene, a specific mRNA site, and a primer combination for amplifying the corresponding site; The specific mRNA sites include HBB, HBA, MSMB, KLK3, CYP2B7P, SPINK5, KRT4, and KRT13; the housekeeping genes include GAPDH and B2M; each set of primers in the primer combination includes two inner primers, two outer primers, and two loop primers, and their specific sequences are shown in SEQ ID NOs. 1 to 60.

2. A primer combination for body fluid detection based on RT-LAMP technology, characterized in that: Each set of primers in the primer combination includes two inner primers, two outer primers and two loop primers, and their specific sequences are shown in SEQ ID NOs. 1 to 60.

3. The primer combination according to claim 2, characterized in that At least one primer in each primer set spans the exon junction region.

4. The primer combination according to claim 2 or 3, characterized in that The body fluid is at least one of blood, semen, saliva and vaginal secretions.

5. A kit, characterized in that Comprising the primer combination according to any one of claims 2 to 4.

6. Use of the primer combination according to any one of claims 2 to 4, or the kit according to claim 5, in the preparation of reagents for forensic detection and identification.

7. Use of the primer combination according to any one of claims 2 to 4, or the kit according to claim 5, in the preparation of a preparation for detection and identification of body fluids.

8. A method for detecting body fluids based on RT-LAMP technology, characterized in that: The following steps are involved: (1) Treat the sample to be tested with a lysis buffer to obtain RNA from the sample to be tested; (2) Using the primer combination described in any one of claims 2 to 4, or the kit described in claim 5, the reverse transcription loop-mediated isothermal amplification technology is used to amplify the RNA of the sample to be tested, and determine whether the sample contains body fluid components based on the amplification results.

9. The method according to claim 8, characterized in that The amplification system included the following components at the following concentrations: Tris-HCl 50 mM / L, GLC 2.66%, dNTPs 1.2–1.4 mM / L, DT-40 0.83x, MgSO4 6 mM / L, Tween-20 0.10%, HNB 0.06 mM / L, F3 / B3 0.4 μM / L, FIP / BIP 1.6–3.2 μM / L, LF / LB 0.8–1.6 μM / L, Bst 3.0 0.32 U / μL, RI / RT 2 U / μL; The amplification reaction conditions were 65°C and 30 min.

10. The method according to claim 8, characterized in that The lysis buffer includes the following components at the following concentrations: Triton 0.5x, EVA 0.5x, KCl 75-100 mM, KOH 20-25 mM.

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