Multi-target Hantaan disease virus nucleic acid detection kit and detection method

Abstract

The invention discloses a multi-target Hantaan disease virus nucleic acid detection kit and a detection method, and the kit comprises an RT-RPA isothermal amplification reaction system and a CRISPR / Cas12 detection reaction system; the RT-RPA isothermal amplification reaction system is an RT-RPA isothermal amplification primer aiming at the specificity of the hantaan disease virus M gene; the CRISPR / Cas12 detection reaction system is crRNA (Ribonucleic Acid) of the hantavirus, which is specifically recognized by aiming at the CRISPR / Cas12. According to the invention, the CRISPR / Cas12 is combined with a reverse transcription recombinase isothermal amplification (RT-RPA) technology to detect the hantavirus nucleic acid, so that the key limitation problem that the hantavirus nucleic acid PCR detection technology depends on special instruments is effectively solved, the problem that POCT is difficult to realize is solved, and the detection time is shortened.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and in particular relates to a multi-target Hantaan virus nucleic acid detection kit and a detection method. Background Art

[0002] Hemorrhagic fever with renal syndrome (HFRS), also known as epidemic hemorrhagic fever, is a natural epidemic disease caused by Hantavirus with rodents as the main source of infection. Hantavirus belongs to the Bunyavirales order and the Hantaviridae family, and is round or oval in shape, with an average diameter of 120nm. It has a lipid outer membrane and a single-stranded negative-strand RNA genome (divided into three segments: L, M, and S), which encodes the RNA polymerase, envelope glycoprotein, and nucleocapsid protein of the virus. China is one of the high-incidence areas of hemorrhagic fever, with Shaanxi and Heilongjiang being the main epidemic areas. Epidemiological data show that the annual mortality rate of HFRS fluctuates between 0.60% and 13.97%, with a total mortality rate of 2.86%. The latent infection rate in epidemic areas can reach 3.5% to 33%, showing a high incidence in spring and autumn and winter. However, the current detection methods for Hantavirus include PCR method, antigen-antibody detection method, and etiology detection. None of the above methods can simultaneously meet the requirements of high specificity, good repeatability, rapidity, simplicity, and sensitivity. Among them, 1) Antigen detection method: the preparation of specific antibodies is difficult, and the interpretation of results is subjective, with poor specificity; 2) Antibody detection: Antigen-antibody reaction detection has certain false negatives or false positives, and the detection specificity and sensitivity are low; 3) Nucleic acid detection: It can be used as a method for specific detection of Hantavirus, with high specificity and sensitivity, but there are high requirements for experimental conditions, long detection result cycle (4 to 6 hours), and special instruments and equipment are required. ④ Pathogen detection method: It can assist in the detection of Hantavirus, requiring professional laboratories and professional technicians; limiting its wide application. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a multi-target Hantaan virus nucleic acid detection kit to solve the problem of low sensitivity of existing nucleic acid detection.

[0004] The invention also discloses a multi-target Hantaan disease virus nucleic acid detection method.

[0005] In order to achieve the above-mentioned purpose, the technical scheme of the present invention is implemented as follows: a multi-target Hantaan disease virus nucleic acid detection kit, comprising an RT-RPA isothermal amplification reaction system and a CRISPR / Cas12 detection reaction system; the RT-RPA isothermal amplification reaction system is a specific RT-RPA isothermal amplification primer for the Hantaan disease virus M gene; the CRISPR / Cas12 detection reaction system is a crRNA for the Hantaan virus specifically recognized by CRISPR / Cas12.

[0006] Preferably, the specific RT-RPA isothermal amplification primers for the Hantaan virus M gene are: M-RPA-primier-F and M-RPA-primier-R.

[0007] Preferably, the nucleic acid primer sequence of the M-RPA-primier-F is: 5`-GTCACTTTGGAGACCCTGGTGATGTTATGGGTC-3`; the nucleic acid primer sequence of the M-RPA-primier-R is: 5`-GTGTCACACTTTCTGCACCATAACAAATTGCC-3`.

[0008] Preferably, the Hantavirus crRNA includes crRNA-4, crRNA-5, crRNA-6, crRNA-29, crRNA-30, and crRNA31.

[0009] Preferably, the crRNA-4 sequence is: 5`-GCCGAAAATCCTTGTAAAGT-3`; the crRNA-5 sequence is: 5`-AGGCTATAAGAAAGTTCTTG-3`; the crRNA-6 sequence is: 5`-ACTGATGAGAGAATTGAATG-3`; the crRNA-29 sequence is: 5`-GAAATAACAATATTGATATG-3`; the crRNA-30 sequence is: 5`-TGAATATGATGGGAATATTA-3`; the crRNA-31 sequence is: 5`-ACACAAGCAATATACATTTC-3`.

[0010] Another technical solution of the present invention is achieved as follows: a multi-target Hantaan virus nucleic acid detection method, the detection method is specifically:

[0011] S1. Hantavirus gene search: Download the nucleic acid sequence of Hantavirus through the Nucleotide sub-library of a specific database

[0012] S2. Conservative sequence analysis: MAG was used to perform conservative analysis on the downloaded nucleic acid sequence of the Hantavirus, and the conservative sequences of the L, M and S nucleic acid regions were obtained respectively. After data analysis, RT-RPA primers were designed for the M fragment; the RT-RPA primers designed for the M fragment were M-RPA-primier-F or M-RPA-primier-R;

[0013] S3. Design of a specific crRNA sequence for the Hantavirus M fragment: After obtaining the nucleic acid sequence of the M fragment, analyze and obtain the conserved sequence of the nucleic acid region of the M fragment, use software to design crRNA, perform specific comparison analysis on the designed crRNA, and obtain multi-target crRNA.

[0014] Preferably, in S1, the specific database is the Nucleotide sub-library of the NCBI database.

[0015] Compared with the prior art, the present invention combines CRISPR / Cas12 with reverse transcriptase recombinase isothermal amplification (RT-RPA) technology to detect Hantavirus nucleic acid, effectively solving the key limitation problem of dependence on special instruments in the process of Hantavirus nucleic acid PCR detection technology, as well as the difficulty in realizing on-site detection (POCT), thereby shortening the detection time; secondly, the present invention optimizes the detection system by designing a mixed detection reaction system of multi-target crRNA, thereby optimizing and improving the problem of low sensitivity of CRISPR / Cas12 detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of a multi-target Hantaan virus nucleic acid detection kit and the principle of the detection method;

[0017] FIG2 is a schematic diagram of the fluorescence detection results after the RT-RPA product is added to the CRISPR / Cas12 detection reaction system for reaction; wherein:

[0018] Figure 2A The fluorescence detection results are shown in Figure 2. Figure 1 ;

[0019] Figure 2B This is the second schematic diagram of the fluorescence detection results;

[0020] Figure 2C The fluorescence detection results are shown in Figure 2. Figure 3 ;

[0021] Figure 2D The fluorescence detection results are shown in Figure 2. Figure 4 ;

[0022] Figure 3This is a schematic diagram of the detection results under UV light after the RT-RPA product is added to the CRISPR / Cas12 detection reaction system for reaction;

[0023] Figure 4 Schematic diagram of the RNA concentration results detected by the test strip after the RT-RPA product is added to the CRISPR / Cas12 detection reaction system for reaction. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] CRISPR / Cas technology is widely used as a new gene editing technology. However, with the deepening of research, it was found that CRISPR / Cas12 and CRISPR / Cas13 activated their nonspecific cutting activity after specifically recognizing the target sequence under the guidance of guide RNA (gRNA), and their nonspecific cutting reaction system nucleic acid sequence. However, the detection sensitivity of the CRISPR / Cas system is low without in vitro amplification. Therefore, it is necessary to first amplify the detected target sequence in vitro to improve the detection sensitivity. The target sequence recognized by CRISPR / Cas13 is RNA, which increases the detection steps and its RNA has high requirements for environmental conditions. Therefore, CRISPR / Cas12 is combined with reverse transcriptase recombinase isothermal amplification (RT-RPA) technology to achieve a major breakthrough in the rapid nucleic acid detection using the CRISPR / Cas system, reducing the key limitation of nucleic acid detection on instrumentation. This establishes RPA combined with CRISPR / Cas12 technology to detect Hantavirus, improve the ability of nucleic acid detection, and has the advantages of sensitivity, simplicity, accuracy and speed.

[0026] An embodiment of the present invention provides a multi-target Hantaan virus nucleic acid detection kit based on RT-RPA and CRISPRCas12, comprising an RT-RPA isothermal amplification reaction system and a CRISPR / Cas12 detection reaction system; the RT-RPA isothermal amplification reaction system is a specific RT-RPA isothermal amplification primer for the Hantaan virus M gene; the CRISPR / Cas12 detection reaction system is a crRNA for the Hantaan virus specifically recognized by CRISPR / Cas12.

[0027] In the specific implementation process of the embodiment of the present invention, the specific RT-RPA isothermal amplification primers of the Hantaan virus M gene are: M-RPA-primier-F and M-RPA-primier-R.

[0028] In the specific implementation process of the embodiment of the present invention, the M-RPA-primier-F nucleic acid primer sequence is: 5`-GTCACTTTGGAGACCCTGGTGATGTTATGGGTC-3`; the M-RPA-primier-R nucleic acid primer sequence is: 5`-GTGTCACACTTTCTGCACCATAACAAATTGCC-3`.

[0029] In the specific implementation process of the embodiment of the present invention, the Hantavirus criRNA includes crRNA-4, crRNA-5, crRNA-6, crRNA-29, crRNA-30, and crRNA31.

[0030] In the specific implementation process of the embodiment of the present invention, the crRNA-4 sequence is: 5`-GCCGAAAATCCTTGTAAAGT-3`; the crRNA-5 sequence is: 5`-AGGCTATAAGAAAGTTCTTG-3`; the crRNA-6 sequence is: 5`-ACTGATGAGAGAATTGAATG-3`; the crRNA-29 sequence is: 5`-GAAATAACAATATTGATATG-3`; the crRNA-30 sequence is: 5`-TGAATATGATGGGAATATTA-3`; the crRNA-31 sequence is: 5`-ACACAAGCAATATACATTTC-3`.

[0031] The embodiment of the present invention also provides a multi-target Hantaan virus nucleic acid detection method based on RT-RPA and CRISPR / Cas12d, and the detection method is specifically as follows:

[0032] S1. Hantavirus gene search: Download the nucleic acid sequence of Hantavirus through the Nucleotide sub-library of a specific database

[0033] S2. Conservative sequence analysis: MAG was used to perform conservative analysis on the downloaded nucleic acid sequence of the Hantavirus, and the conservative sequences of the L, M and S nucleic acid regions were obtained respectively. After data analysis, RT-RPA primers were designed for the M fragment; the RT-RPA primers designed for the M fragment were M-RPA-primier-F or M-RPA-primier-R;

[0034] S3. Design of a specific crRNA sequence for the Hantavirus M fragment: After obtaining the nucleic acid sequence of the M fragment, analyze and obtain the conserved sequence of the nucleic acid region of the M fragment, use software to design crRNA, perform specific comparison analysis on the designed crRNA, and obtain multi-target crRNA.

[0035] In a specific implementation process of the embodiment of the present invention, in S1, the specific database is the Nucleotide sub-library of the NCBI database.

[0036] The following are specific embodiments

[0037] The present invention provides a design strategy detection combination for detecting Hantavirus nucleic acid combination based on CRISPR / Cas12 combined with recombinase isothermal amplification (RT-RPA) technology, including a constant temperature amplification reaction system of RT-RPA and a detection reaction system of CRISPR / Cas12. The RT-RPA amplification reaction system specifically targeting the M gene of Hantavirus isothermal amplification primers, M-RPA-primier-F: M-RPA-primier-R;

[0038] Wherein, the sequence of the M-RPA-primier-F nucleic acid primer is:

[0039] 5`-GTCACTTTGGAGACCCTGGTGATGTTATGGGTC-3`,

[0040] The sequence of the M-RPA-primier-R nucleic acid primer is:

[0041] 5`-GTGTCACACTTTCTGCACCATAACAAATTGCC-3`;

[0042] A detection reaction system for CRISPR / Cas12, including criRNA of Hantavirus specifically recognized by CRISPR / Cas12 and a reaction system.

[0043] The specific crRNAs designed include crRNA-4, crRNA-5, crRNA-6, crRNA-29, crRNA-30, and crRNA31.

[0044] Among them, the crRNA-4 sequence is: 5`-GCCGAAAATCCTTGTAAAGT-3`;

[0045] The sequence of crRNA-5 is: 5`-AGGCTATAAGAAAGTTCTTG-3`;

[0046] The sequence of crRNA-6 is: 5`-ACTGATGAGAGAATTGAATG-3`;

[0047] The crRNA-29 sequence is: 5`-GAAATAACAATATTGATATG-3`;

[0048] The sequence of crRNA-30 is: 5`-TGAATATGATGGGAATATTA-3`;

[0049] The crRNA-31 sequence is: 5`-ACACAAGCAATATACATTTC-3`.

[0050] Furthermore, the detection fluorescent probe is a ssDNA fluorescent probe, the fluorescent probe sequence is 5'-FAM- and 3'BHQ1, or the 5' labeling molecule of the above probe sequence is modified to biotin to obtain a colloidal gold detection probe.

[0051] Furthermore, the CRISPR / Cas12 detection system also includes the following: the ratio of CRISPR / Cas12, crRNA, RT-RPA product and other reaction systems in the reaction system;

[0052] The present invention provides a multi-target Hantaan virus nucleic acid detection kit and detection method based on RT-RPA and CRISPR / Cas12, which mainly comprises the following steps:

[0053] (1) Hantavirus gene search

[0054] The nucleic acid sequence of Hantavirus was downloaded from the Nucleotide sub-database of NCBI (https: / / www.ncbi.nlm.nih.gov / );

[0055] (2) Conserved sequence analysis

[0056] MAG was used to perform conservation analysis on the nucleic acid sequences of all downloaded Hantaviruses, and the conserved sequences of the L, M, and S nucleic acid regions were obtained. After data analysis, RT-RPA primers (M-RPA-primier-F / M-RPA-primier-R) were designed for the M segment.

[0057] (3) Design of crRNA sequence specific for Hantavirus M segment

[0058] After obtaining the nucleic acid sequence of the M fragment, the conservative sequence of the nucleic acid region of the M fragment was analyzed and obtained, and the crRNA was designed using the http: / / crispr-era.stanford.edu / index.jsp software, and the designed crRNA was specifically compared and analyzed. The Blast data sub-library in the https: / / www.ncbi.nlm.nih.gov / database was compared and analyzed to obtain the multi-target crRNA. The design principle is shown in the schematic diagram. 1d, and the multi-target crRNA sequence was designed.

[0059] Testing process

[0060] The detection process of the multi-target Hantavirus nucleic acid detection kit and detection method based on RT-RPA and CRISPR / Cas12 is as follows Figure 1 ;

[0061] 1) Extraction of RNA: use a kit to extract RNA from the sample;

[0062] 2) Reverse transcription of the RNA obtained in step 1) is performed using the primers in the RT-RPA reaction system to obtain the target fragment DNA sequence.

[0063] 3) Add the RT-RPA product obtained in step 2) to the CRISPR / Cas12 detection reaction system, react at 37° for 30 min, and detect the fluorescence signal as follows: Figures 2A-2D , or ultraviolet light to observe the signal such as Figure 3 , and the test results are detected by test strips. Figure 4 .

[0064] A multi-target Hantavirus nucleic acid detection kit based on RT-RPA and CRISPR / Cas12, and a reaction system of the CRISPR / Cas12 detection system of Hantavirus, wherein the reaction system includes a 42-degree isothermal amplification reaction system of RT-RPA and a detection reaction system of CRISPR / Cas12. RT-RPA 42-degree isothermal amplification reaction system: Buffer A 29.4μL, 10μM primers of M-RPA-primier-F and M-RPA-primier-R 2μL each, Buffer B 2.5μL, template RNA 5μL, ddH2O 4.1μL total volume, 42 degrees reaction for 30min. The detection reaction system of CRISPR / Cas12 is a total reaction system of 30 μL; Buffer 3 μL, Cas12 enzyme 1 μL, ssDNA probe 6 μL, RT-RPA product 5 μL, crRNA (4 / 5 / 6 / 29 / 30 / 31) 2 μL each, ddH2O 3 μL, total volume 30 μL.

[0065] A multi-target Hantavirus nucleic acid detection kit and detection method based on RT-RPA and CRISPR / Cas12 were obtained, and the fluorescence detection method obtained a detection RNA concentration of 10 -16 M, the test strip shows that the detection RNA concentration is 10 -13 M.

[0066] In summary, the present invention combines CRISPR / Cas12 with RT-RPA technology to detect Hantavirus nucleic acid, effectively solving the key limitation problem of dependence on special instruments in the process of Hantavirus nucleic acid PCR detection technology, as well as the difficulty of realizing on-site detection (POCT), thereby shortening the detection time; secondly, the present invention optimizes the detection system by designing a mixed detection reaction system of multi-target crRNA, thereby optimizing and improving the problem of low sensitivity of CRISPR / Cas12 detection

[0067] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A multi-target Hantaan virus nucleic acid detection kit, characterized in that: It includes an RT-RPA isothermal amplification reaction system and a CRISPR / Cas12 detection reaction system; the RT-RPA isothermal amplification reaction system is a specific RT-RPA isothermal amplification primer for the M gene of the Hantaan disease virus; the CRISPR / Cas12 detection reaction system is a crRNA for the Hantaan virus specifically recognized by CRISPR / Cas12.

2. A multi-target Hantaan virus nucleic acid detection kit according to claim 1, characterized in that: The specific RT-RPA isothermal amplification primers of the Hantaan virus M gene are: M-RPA-primier-F and M-RPA-primier-R.

3. A multi-target Hantaan virus nucleic acid detection kit according to claim 1, characterized in that: The nucleic acid primer sequence of the M-RPA-primier-F is: 5`-GTCACTTTGGAGACCCTGGTGATGTTATGGGTC-3`; the nucleic acid primer sequence of the M-RPA-primier-R is: 5`-GTGTCACACTTTCTGCACCATAACAAATTGCC-3`.

4. A multi-target Hantaan virus nucleic acid detection kit according to claim 1, characterized in that: The Hantavirus crRNA includes crRNA-4, crRNA-5, crRNA-6, crRNA-29, crRNA-30, and crRNA31.

5. A multi-target Hantaan virus nucleic acid detection kit according to claim 4, characterized in that: The crRNA-4 sequence is: 5`-GCCGAAAATCCTTGTAAAGT-3`; the crRNA-5 sequence is: 5`-AGGCTATAAGAAAGTTCTTG-3`; the crRNA-6 sequence is: 5`-ACTGATGAGAGAATTGAATG-3`; the crRNA-29 sequence is: 5`-GAAATAACAATATTGATATG-3`; the crRNA-30 sequence is: 5`-TGAATATGATGGGAATATTA-3`; the crRNA-31 sequence is: 5`-ACACAAGCAATATACATTTC-3`.

6. A multi-target Hantaan virus nucleic acid detection method, characterized in that: The specific detection method is: S1. Hantavirus gene search: Download the nucleic acid sequence of Hantavirus through the Nucleotide sub-library of a specific database S2. Conservative sequence analysis: MAG was used to perform conservative analysis on the downloaded nucleic acid sequence of the Hantavirus, and the conservative sequences of the L, M and S nucleic acid regions were obtained respectively. After data analysis, RT-RPA primers were designed for the M fragment; the RT-RPA primers designed for the M fragment were M-RPA-primier-F or M-RPA-primier-R; S3. Design of a specific crRNA sequence for the Hantavirus M fragment: After obtaining the nucleic acid sequence of the M fragment, analyze and obtain the conserved sequence of the nucleic acid region of the M fragment, use software to design crRNA, perform specific comparison analysis on the designed crRNA, and obtain multi-target crRNA.

7. A multi-target Hantaan disease virus nucleic acid detection method according to claim 6, characterized in that: In S1, the specific database is the Nucleotide sub-library of the NCBI database.

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