Primer probe combination, kit and method for detecting rift valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a

Abstract

The invention discloses a rift valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a detection primer probe combination, a kit and a method, and belongs to the technical field of molecular detection. In order to solve the problems that an existing RVFV detection technology is high in equipment dependence, low in sensitivity, not suitable for primary laboratories and epidemic disease sites and the like in clinical application, a highly conserved sequence of an S gene of an RVFV ZH-548 strain is selected as a detection target through comparative analysis of an RVFV S gene sequence, and the RVFV ZH-548 strain S gene is detected on the basis of the target. A group of primers and probes for RVFV nucleic acid RT-RAA-CRISPR / Cas12a visual detection are researched and developed, a corresponding detection method is established, and a matched detection kit is provided. The detection method provided by the invention is high in sensitivity, strong in specificity, simple and convenient to operate and high in safety, and has remarkable advantages in scenes such as cross-border animal quarantine, epidemic area field emergency detection and grassroots pathogen rapid screening.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of molecular detection, and particularly relates to a Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a detection primer probe combination, a kit and a method. BACKGROUND

[0002] Rift Valley Fever Virus (RVFV) is a single-stranded negative-sense segmented RNA virus belonging to the genus of Phlebovirus in the family of Bunyaviridae. The virus was first discovered in the East African Rift Valley in Kenya in 1931, and since then, more than 30 countries have reported related cases, about 80% of which are concentrated in Africa. Rift Valley Fever (RVF) is an acute viral infection caused by Rift Valley fever virus, which is transmitted by mosquito vectors or contact. Due to its high pathogenicity and transmission risk, RVF is listed in the "priority pathogens" list by the World Health Organization (WHO), and is also identified as a notifiable animal disease by the World Organisation for Animal Health (WOAH). RVFV has a wide spectrum of infection, and can infect humans and livestock animals such as buffalos, camels, goats and sheep. The virus mainly harms young and pregnant animals, among which goat young and lambs are most susceptible, with a mortality rate of 70% to 100%, and the abortion rate of pregnant animals can reach 100%. Humans are mainly infected by contacting the blood of sick animals or eating raw meat of sick animals. After human infection, symptoms such as fever, headache and muscle and joint pain often occur, and in severe cases, encephalitis, hepatitis, blindness and even death can occur.

[0003] The existing RVFV detection methods mainly include virus isolation, enzyme-linked immunosorbent assay, reverse transcription polymerase chain reaction (RT-PCR) and plaque reduction neutralization test, etc. Among them, RT-PCR is widely used in the detection of RVFV due to its mature technical advantages, and is listed by WOAH as the standard method for laboratory diagnosis of RVF. However, due to the need for precise temperature control equipment and long detection period, RT-PCR is not suitable for rapid screening in primary laboratories and disease sites. Virus isolation is the gold standard for diagnosing RVF, but it is not suitable for primary laboratories due to its complicated operation and the need for biological protection facilities. RVFV antibodies are lagging, and usually appear five days after the onset, which is not suitable for early rapid diagnosis. The limitations of these methods limit their widespread application in resource-lacking areas or on-site detection.

[0004] The nucleic acid rapid detection method based on isothermal amplification technology is more suitable for early rapid diagnosis of RVF at the grassroots level. The primer probe set and kit for detecting RVFV by RAA fluorescence method disclosed in Chinese patent application No. CN108624720A can complete the detection within 15 min, but its sensitivity to RVFV recombinant plasmid is 10 copies / μL, which needs to be improved. The RVFV nucleic acid rapid detection system, kit and use method based on RT-RPA and CRISPR / Cas12a disclosed in Chinese patent application No. CN119776589A can detect 1 copies / μL of RVFV recombinant plasmid, which has high sensitivity, but due to the disadvantage of high dependence on a fluorescence quantitative PCR instrument, it cannot meet the demand of on-site detection. Therefore, it is urgent to develop a convenient, easy-to-promote, high-sensitivity RVFV nucleic acid detection method more suitable for grassroots laboratories and epidemic sites to effectively curb the spread of RVF epidemic and help vaccine development. SUMMARY

[0005] The present application aims to solve the problems of high cost, strong equipment dependence, complicated operation process, low sensitivity, and unsuitability for grassroots laboratories and epidemic sites in the clinical application of existing RVFV detection technologies, and provides a nucleic acid visual detection method for RVFV and its matching primer, probe and kit for customs or grassroots laboratories. By comparing and analyzing the gene sequences of 39 strains of RVFV isolated at different times and in different regions published in the GenBank database, the highly conserved sequence (1 bp-459 bp) of RVFV ZH-548 strain (Genebank number: NC_014395.1) is selected as the detection target. Based on the target, the present application develops a set of primers and probes for RVFV nucleic acid RT-RAA-CRISPR / Cas12a visual detection, establishes the corresponding detection method, and provides a matching detection kit. S The highly conserved sequence (1 bp-459 bp) of RVFV ZH-548 strain (Genebank number: NC_014395.1) S The highly conserved sequence (1 bp-459 bp) of RVFV ZH-548 strain (Genebank number: NC_014395.1)

[0006] To solve the above technical problems and achieve the corresponding technical effects, the present application provides the following technical solutions: The first object of the present application is to provide a primer probe combination for RVFV nucleic acid RT-RAA-CRISPR / Cas12a visual detection, which is composed of gRNA, single-stranded DNA probe and RT-RAA primer pair; wherein the nucleotide sequence of gRNA is shown in SEQ ID NO. 1, the nucleotide sequence of single-stranded DNA probe is TTATT, and the nucleotide sequences of the upper and lower primers of the RT-RAA primer pair are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0007] In one embodiment of the present application, the single-stranded DNA probe is connected with FAM as a reporter group at the 5' end and BHQ1 as a quencher group at the 3' end.

[0008] A second object of the present application is to provide an application of the primer probe combination described above in the visual detection of Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a for non-disease diagnosis purposes.

[0009] A third object of the present application is to provide an application of the primer probe combination described above in the preparation of a Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a visual detection kit.

[0010] A fourth object of the present application is to provide a kit for Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a visual detection, which contains the primer probe combination described above.

[0011] In a preferred embodiment of the present application, the kit further comprises a recombinant plasmid pcDNA3.1-RVFV-S as a template, which carries the nucleotide sequence of the RVFV S gene as shown in SEQ ID NO. 4.

[0012] Preferably, the kit further comprises RT-RAA nucleic acid amplification reagents (basic type) and Cas12a protein, 10x Borealis Buffer required for CRISPR / Cas12a reaction; the RT-RAA nucleic acid amplification reagents (basic type) contain the following components: dNTP, single-strand binding protein, RNase Free ddH2O, recombinase, DNA polymerase, reverse transcriptase, magnesium acetate solution.

[0013] A fifth object of the present application is to provide an application of the kit described above in the visual detection of Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a for non-disease diagnosis purposes.

[0014] A sixth object of the present application is to provide a Rift Valley fever virus nucleic acid visual detection method for non-disease diagnosis purposes, which comprises the following steps: S1, taking a sample to be tested, and extracting nucleic acid in the sample by using a nucleic acid extraction reagent; S2, performing RT-RAA amplification on the nucleic acid obtained in S1 by using the RT-RAA primer pair and the reagents required for RT-RAA amplification according to claim 1 to obtain an RT-RAA amplification product; S3, performing CRISPR / Cas12a detection on the RT-RAA amplification product obtained in S2 by using the gRNA, the single-stranded DNA probe and the reagents required for the CRISPR / Cas12a reaction of claim 1; S4, performing result interpretation by using the 490 nm excitation light irradiation device: if no green fluorescence appears in the negative control and green fluorescence appears in the sample, the sample contains Rift Valley fever virus; if no green fluorescence appears in both the negative control and the sample, the sample does not contain Rift Valley fever virus; if green fluorescence appears in both the negative control and the sample, the detection result is invalid.

[0015] In a preferred embodiment of the present application, the reagents required for the RT-RAA amplification in S2 are the RT-RAA nucleic acid amplification kit (basic type) of Hangzhou Zongce Biological Co., Ltd.

[0016] Preferably, the total volume of the RT-RAA amplification system in S2 is 25 μL, and the specific preparation operation is as follows: 25 μL of Buffer A, 2.5 μL of Buffer B, 13.5 μL of RNaseFree ddH2O, 2 μL of each of the upstream and downstream primers (concentration of 10 μmol / L) are mixed, and the premix is transferred to the freeze-dried powder containing the enzymes required for amplification; after mixing uniformly, 20 μL is taken at the bottom of the reaction tube, and 5 μL of the sample to be detected is added.

[0017] Preferably, the RT-RAA amplification in step S2 is combined with the CRISPR / Cas12a detection in S3 by one-tube method operation, and the specific operation is as follows: the CRISPR / Cas12a reaction premix is prepared in the PCR tube, and is embedded in the RT-RAA amplification reaction tube upside down; after the RT-RAA reaction is completed, low-speed centrifugation is performed to mix the CRISPR / Cas12a premix and the amplification product uniformly, and the CRISPR / Cas12a reaction is performed.

[0018] In an embodiment of the present application, the total volume of the CRISPR / Cas12a detection system is 50 μL, which is composed of the following components: 5 μL of Cas12a protein, 5 μL of 10×Borealis Buffer, 2.5 μL of single-stranded DNA probe (concentration of 2 μmol / L), 2.5 μL of gRNA, 10 μL of RNaseFree ddH2O, and 25 μL of RT-RAA amplification product.

[0019] In a preferred embodiment of the present application, the reaction temperature of the RT-RAA amplification in S2 is 39℃, and the reaction time is 30 min; the reaction temperature of the CRISPR / Cas12a detection in S3 is 37℃, and the reaction time is 20 min.

[0020] In a preferred embodiment of the present application, the working concentrations of Cas12a protein and gRNA in the reaction system for CRISPR / Cas12a detection described in S3 are 0.053 μmol / L and 0.075 μmol / L, respectively.

[0021] A seventh object of the present application is to provide the application of the above-mentioned RVFV nucleic acid visual detection method in the existence screening of RVFV epidemic tracing or non-diagnostic purposes.

[0022] In the RVFV detection scenario, in addition to disease diagnosis and treatment, there are also various non-diagnostic purposes for detection needs, such as entry-exit animal quarantine supervision, virus detection of vector organisms, etc. The above-mentioned primer probe combination, kit and detection method can be applied to RVFV epidemiological research, RVFV epidemic transmission path tracking, or non-diagnostic purposes for RVFV existence screening of the samples to be detected (such as animal organ specimens, blood products, insect grinding specimens, environmental swab specimens, etc.).

[0023] The beneficial effects of the present application are: (1) The RVFV nucleic acid RT-RAA-CRISPR / Cas12a visual detection method established by the present application has high sensitivity, and the minimum detectable recombinant plasmid is 0.5 copies / μL within 50 min. Compared with the real-time fluorescent RT-PCR method recommended by WOAH (detection limit 4.3-8 copies / μL), the present application shortens the detection period, simplifies the operation process, reduces the professional requirements for the operator, and improves the sensitivity by nearly 10 times. In addition, the present application uses the trans-cleavage activity of the CRISPR / Cas12a system to cut the single-stranded DNA probe, which can be judged by green fluorescence signal under 490 nm excitation light irradiation without complex statistical analysis.

[0024] (2) The RVFV nucleic acid RT-RAA-CRISPR / Cas12a visual detection method established by the present application has strong specificity, and can accurately detect RVFV vaccine strain MP-12 RNA, and has no cross reaction with EBoV RNA, NiV RNA, SFTFV RNA and LASV recombinant plasmid, indicating that the present application has good anti-interference ability in complex sample detection, and provides reliable technical support for clinical detection.

[0025] (3) The RVFV nucleic acid RT-RAA-CRISPR / Cas12a visual detection method established by the application can be completed by using a constant temperature water bath or a heat preservation cup and a 490 nm excitation light irradiation device without precise temperature control equipment; among many isothermal amplification technologies, the RT-RAA technology with a similar reaction temperature to the CRISPR / Cas12a system is selected, one tube detection is realized, the activity of the Cas12a protease in the detection process is ensured, the detection result is visualized, and the problem of aerosol pollution caused by traditional isothermal amplification technology is solved; in order to avoid the decline of the amplification efficiency of the RT-RAA due to the cutting of the Cas12a protein on the target, the amplification system and the detection system are placed in different spaces in the tube, so that the amplification efficiency and the detection sensitivity are improved. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 It is the operation flow chart of the RVFV nucleic acid RT-RAA-CRISPR / Cas12a visual detection method in embodiment 2. Figure 2 It is the result graph of optimizing the working concentration of Cas12a protein of the RVFV CRISPR / Cas12a detection system in embodiment 3. Figure 3 It is the result graph of optimizing the working concentration of gRNA of the RVFV CRISPR / Cas12a detection system in embodiment 3. Figure 4 It is the result graph of optimizing the RT-RAA reaction temperature of the RVFV in embodiment 3. Figure 4 A in the figure is the result graph of the RT-RAA reaction of the recombinant plasmid with different concentrations at 37 DEG C and then the CRISPR / Cas12a detection, Figure 4 B in the figure is the result graph of the RT-RAA reaction of the recombinant plasmid with different concentrations at 39 DEG C and then the CRISPR / Cas12a detection, Figure 4 C in the figure is the result graph of the RT-RAA reaction of the recombinant plasmid with different concentrations at 42 DEG C and then the CRISPR / Cas12a detection. Figure 5 It is the sensitivity evaluation result graph of the RVFV nucleic acid RT-RAA-CRISPR / Cas12a visual detection method in embodiment 4. Figure 6 It is the specificity evaluation result graph of the RVFV nucleic acid RT-RAA-CRISPR / Cas12a visual detection method in embodiment 4. DETAILED DESCRIPTION

[0027] To further illustrate the technical solutions of the present application, the embodiments will be described in detail in conjunction with the drawings and examples, but the content described should not be regarded as limiting the scope of protection of the present application. It should be noted that: the described embodiments are only part of the embodiments of the application, and other embodiments obtained by those skilled in the art on the basis of the application without creative labor are also within the scope of protection; the experimental methods involved in the embodiments, if not specifically stated, are all conventional techniques in the art or are operated according to the product instructions; the materials, reagents and instruments used, unless otherwise specified, can be purchased through regular commercial channels or prepared according to conventional methods in the art.

[0028] The reagents used in the present application are as follows: RVFV S The recombinant plasmid pcDNA3.1-RVFV-S containing the RVFV gene (Genebank number: NC_014395.1) was purchased from Shengong Biotechnology Co., Ltd. (Shanghai); the RT-RAA nucleic acid amplification kit (basic type) was purchased from Hangzhou Zhongce Biological Technology Co., Ltd.; the Cas12a protein was prepared by the prokaryotic expression system in the laboratory, and the specific operation can be found in the expression and purification method and application of a LbCpf1 protein disclosed in the Chinese patent application with publication number CN117701531A.

[0029] The specific information of the 39 RVFV reference strains involved in the present application is shown in Table 1.

[0030] Table 1 Specific information of 39 RVFV reference strains

[0031] Example 1: Primers and probes for Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a visual detection To solve the problem of CRISPR / Cas12a off-target caused by the evolution and variation of RVFV, the present application integrates RVFV sequence analysis data, existing detection methods and national standards, and performs deep alignment on the whole genome sequence of RVFV, finds that the nucleotide difference of RVFV S The nucleotide difference of the gene is lower than that of other segment genes, and the average molecular evolution rate is also lower, which has high conservation; the sequences of 39 RVFV S genes isolated from different times and different regions published in the GenBank database are aligned and analyzed by using MAFFT version 7 and SnapGene 6.0.2 software, and the RVFV ZH-548 strain (Genebank number: NC_014395.1) is selected as a template to screen SThe conserved fragment (1 bp-459 bp) is used as a detection target, and the reference strain details are shown in Table 1.

[0032] The present application designs a plurality of gRNAs and a single-stranded DNA probe near the PAM (TTTN sequence) site in the conserved sequence, and screens the gRNA that specifically binds to the target and efficiently activates the Cas12a protein through the fluorescence signal intensity, the nucleotide sequence of the screened gRNA is shown as SEQ ID NO. 1, and the nucleotide sequence of the single-stranded DNA probe is TTA TT. According to the RT-RAA primer design principle, the present application designs 6 sets of specific recognition RVFV S RT-RAA primer pairs of genes, and the best RT-RAA amplification primer pair is screened, the nucleotide sequence of the upstream primer RT-RAA-F is shown as SEQ ID NO. 2, and the nucleotide sequence of the downstream primer RT-RAA-R is shown as SEQ ID NO. 3.

[0033] SEQ ID NO. 1: 5'-UAAUUUCUACUAAGUGUAGAUCUGUGAUAUCUGUUGAUUUGCAG-3'; The nucleotide sequence of the single-stranded DNA probe is 5'-[FAM]ttatt[BHQ1]-3', that is, the 5' end of the probe is connected with FAM, and the 3' end is connected with BHQ1; SEQ ID NO. 2: 5'-CCCTAGTGCTTATCAAGTATATCATGGATTAC-3'; SEQ ID NO. 3: 5'-GGAACCTTGTGAAATCACTAAGAGTCATAT-3'.

[0034] Example 2: Establishment of Rift Valley Fever Virus Nucleic Acid RT-RAA-CRISPR / Cas12a Visual Detection Method The present application uses the recombinant plasmid pcDNA3.1-RVFV-S (the RVFV S gene nucleotide sequence is shown as SEQ ID NO. 4) as a template, uses the primers and single-stranded DNA probe provided in Example 1, and provides a Rift Valley Fever Virus nucleic acid RT-RAA-CRISPR / Cas12a visual detection method. The present application adopts one tube operation (the specific operation process is shown as Figure 1The specific operation steps are as follows: 20 μL of the RT-RAA reaction system is prepared in a 1.5 mL tube, 5 μL of the target is added for RT-RAA amplification. In order to avoid aerosol pollution caused by opening the cover, the PCR tube containing the CRISPR / Cas12a reaction system is inverted in the 1.5 mL tube, and then sealed. According to the experimental procedure shown in the figure, RT-RAA amplification and CRISPR / Cas12a detection are carried out in turn. Each variable setting has a negative control group. After the reaction is completed, the result is judged by using the 490 nm excitation light irradiation device. If the negative control tube does not appear green fluorescence, and the sample tube appears green fluorescence, the sample contains Rift Valley fever virus; if the negative control tube and the sample tube do not appear green fluorescence, the sample does not contain Rift Valley fever virus; if the negative control tube and the sample tube both appear green fluorescence, it is suspected that there is aerosol pollution, and the detection result is invalid. Figure 1 The experimental procedure shown in the figure, RT-RAA amplification and CRISPR / Cas12a detection are carried out in turn. Each variable setting has a negative control group. After the reaction is completed, the result is judged by using the 490 nm excitation light irradiation device. If the negative control tube does not appear green fluorescence, and the sample tube appears green fluorescence, the sample contains Rift Valley fever virus; if the negative control tube and the sample tube do not appear green fluorescence, the sample does not contain Rift Valley fever virus; if the negative control tube and the sample tube both appear green fluorescence, it is suspected that there is aerosol pollution, and the detection result is invalid.

[0035] The preparation of the above-mentioned RT-RAA reaction system uses RT-RAA nucleic acid amplification reagent (basic type), and the specific method is as follows: 25 μL of Buffer A, 2.5 μL of Buffer B, 13.5 μL of RNaseFree ddH2O, 2 μL of each of the upstream and downstream primers (concentration of 10 μmol / L) are mixed to obtain a premix, and the premix is transferred to a freeze-dried powder containing the enzymes required for amplification; after mixing uniformly, 20 μL is taken at the bottom of the reaction tube, and 5 μL of the sample to be detected is added.

[0036] The total volume of the above-mentioned CRISPR / Cas12a reaction system is 50 μL, and the specific components are as follows: 5 μL of Cas12a protein, 5 μL of 10×Borealis Buffer, 2.5 μL of single-stranded DNA probe (concentration of 2 μmol / L), 2.5 μL of gRNA, 10 μL of RNaseFree ddH2O, and 25 μL of RT-RAA amplification product.

[0037] SEQ ID NO.4: Example 3: Optimization of Rift Valley Fever Virus Nucleic Acid RT-RAA-CRISPR / Cas12a Visual Detection Method 1. Optimization of Rift Valley Fever Virus CRISPR / Cas12a Detection System The present application uses 5x10 9 copies / μL of recombinant plasmid pcDNA3.1-RVFV-S as a template, uses the gRNA and single-stranded DNA probe provided in Example 1 to optimize the RVFV CRISPR / Cas12a detection system.

[0038] The working concentration of Cas12a protein was set to 0.027 μmol / L, 0.053 μmol / L, and 0.106 μmol / L, respectively, and a 20 μL CRISPR / Cas12a reaction system was prepared. The 20 μL CRISPR / Cas12a reaction system comprises: 2 μL of Cas12a protein, 2 μL of 10x Borealis Buffer, 1 μL of single-stranded DNA probe (concentration of 2 μmol / L), 1 μL of gRNA (concentration of 2 μmol / L), 2 μL of template, and 12 μL of RNaseFree ddH2O. At the same time, a negative control was set up using a plasmid empty vector as a template, and three replicate groups were set up for each test. The reaction tube was placed in a real-time fluorescent quantitative PCR instrument and incubated at 37℃ for 50 min, and the fluorescent signal was automatically read every 2 min. The real-time monitored fluorescence curve was analyzed, and the optimal working concentration of Cas12a protein was selected. The results are shown in Figure 2 When the working concentration of Cas12a protein is 0.053 μmol / L, the read fluorescent signal value is the highest, and the difference in fluorescent signal value produced by the negative control is significant. Therefore, the optimal working concentration of Cas12a protein in the present application is 0.053 μmol / L.

[0039] The optimal Cas12a protein working concentration was selected, and 5x10 9 copies / μL of recombinant plasmid pcDNA3.1-RVFV-S as a template, gRNA working concentration was set to 0.025 μmol / L, 0.05 μmol / L, 0.075 μmol / L, and 0.1 μmol / L, respectively, and a 20 μL CRISPR / Cas12a reaction premix was prepared according to the above reaction system. A negative control was set up using a plasmid empty vector as a template, and three replicate groups were set up for each test. The reaction tube was placed in a real-time fluorescent quantitative PCR instrument and incubated at 37℃ for 50 min, and the fluorescent signal was automatically read every 2 min. The real-time monitored fluorescence curve was analyzed, and the optimal working concentration of gRNA was selected. The results are shown in Figure 3As shown in Table 1, when the working concentration of gRNA was 0.075 μmol / L, the read fluorescence signal value was the highest, and the fluorescence signal value produced by the negative control was significantly different. Therefore, 0.075 μmol / L was selected as the optimal working concentration of gRNA of the present application.

[0040] 2. Optimization of Rift Valley fever virus RT-RAA reaction conditions The recombinant plasmid pcDNA3.1-RVFV-S diluted by ten times in series was used as the template, the reaction system of the RT-RAA-CRISPR / Cas12a nucleic acid visualization detection method was prepared according to the description in Example 2, and the optimization of the RT-RAA reaction temperature and reaction time was sequentially performed.

[0041] The recombinant plasmid pcDNA3.1-RVFV-S diluted by ten times in series (5×10 -1 copies / μL~5×10 1 copies / μL) was used as the template, the RT-RAA reaction system was placed at 37℃, 39℃, 42℃ for amplification for 30 min, and then incubated at 37℃ for 20 min for CRISPR / Cas12a detection. The plasmid empty vector was used as the template to set up the negative control. The fluorescence signal intensity produced under different temperature conditions was analyzed and compared to screen the optimal amplification temperature of the RT-RAA reaction. The results are shown in Table 1. Figure 4 As shown in Table 1, when the amplification temperature was set to 39℃, the fluorescence signal intensity produced by the reaction tube was the highest, and the detection limit was the lowest. Therefore, the optimal amplification temperature of the RT-RAA of the present application was determined to be 39℃.

[0042] Under the optimal RT-RAA amplification temperature, the recombinant plasmid pcDNA3.1-RVFV-S diluted by ten times in series (5×10 - 2 copies / μL~5×10 1 copies / μL) was used as the template, the reaction system of the RT-RAA-CRISPR / Cas12a nucleic acid visualization detection method was prepared according to the description in Example 2, the RT-RAA reaction system was placed at 39℃, and was amplified for 10 min, 20 min, 30 min, and 40 min, respectively, and then incubated at 37℃ for 20 min for CRISPR / Cas12a reaction. The plasmid empty vector was used as the template to set up the negative control. The fluorescence signal intensity produced after different amplification times was analyzed and compared to screen the optimal amplification time of the RT-RAA. The results are shown in Table 2, and the recombinant plasmid of 0.5 copies / μL could be detected when the RT-RAA was amplified for 30 min and 40 min. In order to shorten the detection time, 30 min was selected as the optimal amplification time of the present application.

[0043] Table 2 Optimization of RT-RAA amplification time

[0044] 3. Evaluation of Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a visual detection method (1) Sensitivity evaluation The recombinant plasmid pcDNA3.1-RVFV-S was diluted by ten times in series, and the concentration range was 5×10 -2 copies / μL~5×10 1 copies / μL of the recombinant plasmid as a template, the sensitivity of the Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a visual detection method optimized in Example 3 was evaluated. At the same time, the plasmid empty vector was used as a template to set up a negative control. The results are shown in Figure 5 When the concentration of the recombinant plasmid is as low as 0.5 copies / μL, a clear yellow-green fluorescence can still be seen in the reaction tube, and the negative control group has no fluorescence. Therefore, the sensitivity of the Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a visual detection method is 0.5 copies / μL of the recombinant plasmid. S

[0045] (2) Specificity evaluation Inactivated veterinary vaccine strain RVFV MP-12 RNA, Nipah virus (NiV) RNA, Lassa fever virus (LASV) N gene recombinant plasmid, Ebola virus (EBoV) RNA, and Severe Fever with Thrombocytopenia Syndrome bunyavirus (SFTFV) RNA were used as templates, and sterile water was used as a negative control. The specificity of the Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a visual detection method optimized in Example 3 was evaluated. The results are shown in Figure 6 The Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a visual detection method can detect RVFV vaccine strain MP-12 RNA, and has no cross-reaction with EBoV RNA, NiV RNA, SFTFV RNA, and LASV recombinant plasmid, indicating that the Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a visual detection method has high specificity and is expected to be applied to clinical detection.

[0046] Example 4: Assembly of Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a visual detection kit ​The gRNA (SEQ ID NO. 1) in Example 1, the single-stranded DNA probe (nucleotide sequence is TTATT), RT-RAA-F (SEQ ID NO. 2), RT-RAA-R (SEQ ID NO. 3) and the recombinant plasmid pcDNA3.1-RVFV-S (carrying the nucleotide sequence of RVFV S gene as shown in SEQ ID NO. 4) in Example 2, RT-RAA nucleic acid amplification reagent (basic type), CRISPR / Cas12a reaction component (containing Cas12a protein, 10x Borealis Buffer) and reaction tube are assembled into Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a visual detection kit.

[0047] Example 5: Application of primers and probes in Example 1, the optimized method in Example 3 and the kit in Example 4 in the detection of Rift Valley fever virus In the scene of Rift Valley fever virus detection, in addition to disease diagnosis and treatment, there are also many detection needs for non-diagnosis and treatment purposes, such as entry-exit animal quarantine supervision, virus detection of disease vectors, etc. Based on this, using Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a visual detection method, the primers and probes recorded in Example 1, the optimized method in Example 3 or the kit described in Example 4 can be applied to Rift Valley fever virus epidemiological research, Rift Valley fever epidemic transmission path tracking, or non-diagnosis and treatment purposes Rift Valley fever virus existence screening carried out for samples to be detected (such as animal organ specimens, blood products, insect grinding specimens, environmental swab specimens, etc.).

[0048] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application. Therefore, the protection scope of the present application should be defined by the claims.

Claims

1. A primer-probe combination for visual detection of Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a, characterized in that: The primer-probe combination consists of gRNA, a single-stranded DNA probe and an RT-RAA primer pair; the nucleotide sequence of the gRNA is shown in SEQ ID NO.1, the nucleotide sequence of the single-stranded DNA probe is TTATT, and the nucleotide sequences of the upstream and downstream primers of the RT-RAA primer pair are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

2. The primer-probe combination according to claim 1, characterized in that The 5' end of the single-stranded DNA probe is connected to FAM as a reporter group, the 3' end is connected to BHQ1 as a quencher group, and the middle core sequence TTATT is complementary to the conserved region of the RVFV S gene and is located near the PAM site.

3. Use of the primer-probe combination according to any one of claims 1 or 2 in the visualization detection of Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a for non-disease diagnosis purposes.

4. Use of the primer-probe combination according to any one of claims 1 or 2 in the preparation of a Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a visualization detection kit.

5. A kit for visual detection of Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a, characterized in that: The kit contains the primer-probe combination according to any one of claims 1 or 2.

6. Use of the kit according to claim 5 in the visual detection of Rift Valley fever virus nucleic acid RT-RAA-CRISPR / Cas12a for non-disease diagnosis purposes.

7. A method for visual detection of Rift Valley fever virus nucleic acid for non-disease diagnosis purposes, characterized in that: The steps include: S1. Take a sample to be tested and extract nucleic acid from the sample using a nucleic acid extraction reagent; S2. Perform RT-RAA amplification on the nucleic acid obtained in S1 using the RT-RAA primer pair and reagents required for RT-RAA amplification according to claim 1 to obtain an RT-RAA amplification product; S3. Perform CRISPR / Cas12a detection on the RT-RAA amplification product obtained in S2 using the gRNA, single-stranded DNA probe, and reagents required for the CRISPR / Cas12a reaction according to claim 1; S4. Use a 490 nm excitation light irradiation device to interpret the results: if the negative control does not show green fluorescence, but the sample shows green fluorescence, the sample contains Rift Valley fever virus; if both the negative control and the sample do not show green fluorescence, the sample does not contain Rift Valley fever virus; if both the negative control and the sample show green fluorescence, the test result is invalid.

8. The method for visual detection of Rift Valley fever virus nucleic acid according to claim 7, characterized in that: The reaction temperature for RT-RAA amplification described in S2 was 39°C, and the reaction time was 30 min; the reaction temperature for CRISPR / Cas12a detection described in S3 was 37°C, and the reaction time was 20 min.

9. The method for visual detection of Rift Valley fever virus nucleic acid according to claim 7, characterized in that: The working concentrations of Cas12a protein and gRNA in the reaction system of CRISPR / Cas12a detection described in S3 were 0.053 μmol / L and 0.075 μmol / L, respectively.

10. Use of the Rift Valley fever virus nucleic acid visualization detection method according to any one of claims 7 to 9 in tracing the source of the Rift Valley fever virus epidemic or screening for the presence of the virus for non-diagnostic purposes.

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