Rapid detection system and kit for nucleic acid of virulent virus based on RT-RPA and CRISPR / Cas12a and use method of rapid detection system and kit

Abstract

The invention discloses an RT-RPA (reverse transcription recombinase polymerase amplification) and CRISPR (clustered regularly interspaced short palindromic repeats) / Cas12a (CRISPR / Cas12a) based rapid detection system and a kit and a use method of the kit. The virulent virus is a Congo fever virus, a Rift Valley fever virus, a Nipah virus or a Hendea virus; the detection system comprises an RT-RPA isothermal amplification system and a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas12a trans-cleavage detection system. The RT-RPA isothermal amplification system comprises an RT-RPA forward primer and an RT-RPA reverse primer which are designed on the basis of viral nucleic acid; the CRISPR / Cas12a trans-cleavage detection system comprises a crRNA sequence which is designed on the basis of viral nucleic acid. The kit comprises the RT-RPA isothermal amplification system and a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas12a (CRISPR / Cas12a) trans-cleavage detection system. The four virulent virus nucleic acids are detected on the basis of RT-RPA and CRISPR / Cas12a systems for the first time, and the kit has the advantages of being high in specificity, high in sensitivity, simple, rapid, independent of complex large instruments and the like, and can be widely applied to clinical sample detection.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a rapid detection system and kit for virulent viral nucleic acid based on RT-RPA and CRISPR / Cas12a. Background Art

[0002] Congo fever virus (Crimean Congo hemorrhagic fever virus, CCHFV), also known as Crimean-Congo hemorrhagic fever virus, is a virulent virus transmitted by ticks, belonging to the order Bunyavirales, family Nairoviridae, genus Orthonairovirus. It is highly contagious and mainly forms a transmission chain from ticks → animals → humans, and ticks → humans → humans. Human infection will show severe bleeding and clinical symptoms of multi-organ damage, mainly headache, fever, vomiting, bleeding, shock and other symptoms, which can lead to death in severe cases, with a case fatality rate of 10-40%. At present, the laboratory diagnosis of Congo fever virus mainly includes serological diagnosis, molecular biological methods and virus culture. Serological detection techniques mainly include indirect immunofluorescence, enzyme-linked immunosorbent assay and immunochromatography. However, the indirect immunofluorescence method has certain requirements for laboratory conditions, and the operation process is relatively cumbersome and requires professional personnel; the enzyme-linked immunosorbent assay requires specific equipment, and the operation process needs to be strictly controlled to avoid false positive or false negative results; the immunochromatographic technology has low sensitivity and specificity. Molecular biology detection technology mainly includes real-time fluorescence quantitative PCR and nucleic acid sequencing, which require expensive and sophisticated equipment, and the operation process is complicated, costly, and time-consuming, and is not suitable for rapid clinical diagnosis; and virus culture has high requirements for laboratory conditions and is time-consuming.

[0003] Rift Valley fever virus (RVFV) is a single-stranded negative-sense RNA virus belonging to the order Bunyavirales, family Leucoviridae, genus Phlebovirus. Rift Valley fever (RVF) is an acute, severe zoonotic infectious disease caused by the Rift Valley fever virus, with mosquitoes as the main transmission medium. The World Organization for Animal Health lists it as a notifiable infectious disease, which must be studied in a biosafety level III laboratory. my country lists it as a Class I infectious disease. RVF can be transmitted through mosquito bites, direct contact, and aerosols, and there is a risk of it being used as a biological warfare agent. The diagnostic methods for Rift Valley fever virus mainly include general examinations, serological tests, etiological tests, and molecular diagnostic techniques. General examinations, such as routine blood tests and urine tests, have low specificity and cannot directly confirm Rift Valley fever virus infection; serological tests mainly determine whether the patient is infected with the virus by detecting specific antibodies in the patient's serum, but it takes a certain amount of time for antibodies to be produced, so antibodies may not be detected in the early stages of infection; etiological testing includes methods such as virus isolation and nucleic acid testing. Virus isolation has high accuracy, but the operation is complex, time-consuming, and has high requirements for laboratory conditions; nucleic acid testing has the advantages of high sensitivity and strong specificity, however, it has high requirements for sample quality and requires professional equipment; molecular diagnostic techniques include polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qPCR), gene sequencing and other technologies, which have the advantages of high sensitivity, strong specificity, and simple operation, but their disadvantages are that they have high requirements for sample quality, require professional equipment and technicians to operate, and are costly.

[0004] Nipah virus (NiV) is a highly pathogenic zoonotic virus of the Paramyxoviridae family. It is an RNA virus and is classified as a biosafety level 4 virus. Its natural host is bats (especially fruit bats), and its intermediate hosts include susceptible animals such as pigs, horses, cats, and dogs. At the same time, NiV has the ability to spread from person to person. After human infection, it usually manifests as symptoms of acute encephalitis and respiratory diseases, with a high mortality rate. The laboratory diagnostic methods of Nipah virus are mainly divided into serological diagnostic methods and molecular diagnostic methods. Traditional serological diagnostic methods mainly include enzyme-linked immunosorbent assay and serum neutralization test.

[0005] Hendra virus (HeV), formerly known as equine measles virus, is a highly lethal zoonotic virus and is classified as a biosafety level 4 virus. It is classified as a Paramyxoviridae and Henipavirus genus. Compared with other Paramyxoviridae viruses, HeV has a wide host range, no hemagglutination and no neuraminidase activity. Fruit bats of the Pteropus family are considered to be the natural hosts of HeV, and they do not show symptoms of disease when infected or carrying the virus. Human infection with HeV can cause fatal respiratory or encephalitis and extensive vascular inflammation. According to surveys, its mortality rate can reach 40-75%. Currently, commonly used laboratory diagnostic techniques for HeV include: etiological detection, immunological detection and molecular biological detection. Initially, serum neutralization experiments were used for preliminary monitoring of HeV, but most infected animals died before an effective immune response was produced, so this method is not suitable for the early detection of HeV infection. Etiological diagnosis is the gold standard for clinical diagnosis of diseases, and isolation and cultivation of HeV from infected animals can assist diagnosis to the greatest extent. However, due to the limitations of HeV on laboratory infrastructure, high operator proficiency, slow and difficult pathogen isolation, etc., it is difficult to use virus isolation and culture methods to meet clinical testing needs in many regions. ELISA is low-cost, highly specific, and highly sensitive, and is widely used in clinical testing, but its specificity is low and leads to frequent false positives and non-specific reactions.

[0006] The CRISPR / Cas system consists of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas). The CRISPR / Cas system in nature is divided into two major categories (category 1 and category 2) based on its structural characteristics and genetic content. These categories are further divided into six types (I to VI) and 33 subtypes based on the function, mode of action and structure of the effector protein. In 1987, the CRISPR sequence structure was first discovered; subsequently, researchers found that the system appeared in more than 50% of bacteria and 90% of archaea. With the deepening of research, the system also has great potential in the field of analytical detection. It is used for analytical detection and has the advantages of identifying targets at physiological temperature, good specificity, high sensitivity, simple operation, fast response speed, low cost, easy miniaturization and integration, etc. It is particularly suitable for low-abundance target molecule detection and POCT diagnostic deployment in complex actual samples. Among them, CIRSPR / Cas3, CRISPR / Cas9, CRISPR / Cas12, CRISPR / Cas13 and CRISPR / Cas14 systems are common systems used for analysis and detection. Usually, Cas12a protein is used for double-stranded DNA detection, Cas13 protein is used for RNA detection, and Cas14 protein is used for single-stranded DNA detection.

[0007] Recombinase polymerase amplification (RPA) has been applied outside the laboratory, in remote areas, and in onboard automated samples because of its convenient detection, high sensitivity, and commercial availability of lyophilized reagents. The RPA reaction principle begins with the binding of the recombinase to the primer, and with the assistance of the loading factor, a nucleoprotein filament is formed. The formed complex searches for the target homologous sequence in the double-stranded DNA. Once the target homologous sequence is located, the complex invades the double-stranded DNA, forming a D-loop structure to initiate the chain exchange reaction, while the unwound chain is stabilized by the single-stranded binding protein. Once the chain exchange is carried out, the recombinase will be disassembled from the nucleoprotein filament and can be used for the next pair of primers. Next, the DNA polymerase extends from the 3' end of the primer. As the amplification continues, the two parental chains begin to separate and eventually form two more parental chains, and then the whole process is repeated.

[0008] The combination of CRISPR / Cas system and RPA amplification to establish a nucleic acid detection method can improve sensitivity and specificity, thereby meeting the needs of rapid, sensitive and simple clinical rapid detection. At present, there are no reports on the use of RT-RPA and CRISPR / Cas12a technology to detect and diagnose the above four virulent viruses. Summary of the invention

[0009] In order to overcome the shortcomings of existing detection technologies for four virulent viruses, the present invention provides a rapid detection system, kit and method for using the virulent virus nucleic acid based on RT-RPA and CRISPR / Cas12a, which can realize the reverse transcription isothermal amplification of the four virulent virus RNA and the integrated visual detection of CRISPR / Cas12a.

[0010] To achieve its purpose, the present invention adopts the following technical solution:

[0011] The present invention provides a rapid detection system for virulent viral nucleic acid based on RT-RPA and CRISPR / Cas12a, wherein the virulent virus is Congo fever virus, Rift Valley fever virus, Nipah virus or Hendra virus; the detection system comprises an RT-RPA isothermal amplification system and a CRISPR / Cas12a trans-cleavage detection system;

[0012] The RT-RPA isothermal amplification system includes RT-RPA forward primers and reverse primers designed based on viral nucleic acid;

[0013] Wherein, the nucleotide sequence of the forward primer of Congo fever virus RT-RPA is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2;

[0014] The nucleotide sequence of the forward primer of Rift Valley fever virus RT-RPA is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4;

[0015] The nucleotide sequence of the Nipah virus RT-RPA forward primer is shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.6;

[0016] The nucleotide sequence of the Hendra virus RT-RPA forward primer is shown in SEQ ID NO.7, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.8;

[0017] The CRISPR / Cas12a trans-cleavage detection system includes crRNA designed based on viral nucleic acid;

[0018] Wherein, the nucleotide sequence of Congo fever virus crRNA is as shown in SEQ ID NO.9;

[0019] The nucleotide sequence of Rift Valley fever virus crRNA is as shown in SEQ ID NO.10;

[0020] The nucleotide sequence of Nipah virus crRNA is as shown in SEQ ID NO.11;

[0021] The Hendra virus crRNA nucleotide sequence is as shown in SEQ ID NO.12.

[0022] As a further preferred embodiment of the technical solution of the present invention, the CRISPR / Cas12a trans-cleavage detection system contains 2 μL of crRNA (500 nM) and further includes:

[0023] Lysis buffer (10X) 3 μL; LbCas12a nuclease (1 μM) 1 μL; ssDNA fluorescent reporter gene (2 μM) 6 μL; DNA template 2 μL; DEPC-H2O 16 μL.

[0024] Preferably, the RT-RPA isothermal amplification system contains 1 μL of Forward primer (10 μM), 1 μL of Reverse primer (10 μM), and further comprises:

[0025] Rehydration buffer (2X) 10μL; RNA template 2μL; ddH2O 4μL; Starter (10X) 2μL.

[0026] The present invention provides a rapid detection kit for virulent viral nucleic acid based on RT-RPA and CRISPR / Cas12a, comprising the above-mentioned RT-RPA isothermal amplification system and CRISPR / Cas12a trans-cleavage detection system.

[0027] The method for using the above-mentioned RT-RPA and CRISPR / Cas12a-based rapid viral nucleic acid detection kit comprises the following steps:

[0028] S1: Add the RNA of the sample to be tested into the RT-RPA amplification system for RT-RPA isothermal amplification to obtain the RT-RPA amplification product;

[0029] S2: Add the RT-RPA amplification product to the CRISPR / Cas12a detection system for reaction;

[0030] S3: Real-time detection of the fluorescence changes of the CRISPR / Cas12a detection system in S2, and drawing of a fluorescence curve; in the drawn fluorescence curve, the detection result whose fluorescence value increases with time is judged as positive, and the result whose fluorescence value is a horizontal line with time is considered as negative.

[0031] Preferably, in step S1, the RT-RPA isothermal amplification conditions are: 39° C. water bath reaction for 30 min.

[0032] Preferably, in step S2, the reaction conditions are: preheating at 25°C for 1 min, 1 cycle; reacting at 37°C for 30 s, 60 cycles, and collecting the fluorescence signal of the FAM fluorescence channel once in each cycle.

[0033] Compared with the prior art, the present invention has the following technical effects:

[0034] 1. Based on RT-RPA and CRISPR / Cas12a detection system, the present invention realizes the rapid detection of nucleic acids of four virulent viruses including Congo fever virus, Rift Valley fever virus, Nipah virus and Hendra virus, and has good specificity and extremely high sensitivity.

[0035] 2. The virulence virus nucleic acid rapid detection system based on RT-RPA and CRISPR / Cas12a provided by the present invention, as well as the detection kit formed based on the detection system, can complete nucleic acid extraction and detection in 1 hour under a constant temperature environment, does not rely on large-scale laboratory instruments and equipment, has a short process and low cost, and is suitable for large-scale promotion and application.

[0036] 3. The present invention provides a rapid detection system and kit for virulent viral nucleic acid based on RT-RPA and CRISPR / Cas12a. The detection results can be reflected by fluorescence values, which is convenient and fast, and is conducive to clinical and grassroots application and popularization. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The results of enzyme digestion identification of the N gene recombinant of Congo fever virus, Rift Valley fever virus, Nipah virus and Hendra virus;

[0038] Figure 2 The results of the expression and identification of N protein of Congo fever virus, Rift Valley fever virus, Nipah virus and Hendra virus;

[0039] Figure 3 , 4 , 5 is the verification of crRNA trans-cleavage function;

[0040] Figure 6 , 7 Results of primer screening for RT-RPA;

[0041] Figure 8 Optimize the reaction temperature for RT-RPA;

[0042] Fig. 9 Results for CRISPR / Cas12a reaction temperature optimization;

[0043] Fig.10 , 11The results of RT-RPA-CRISPR / Cas12a-specific detection of rVSV recombinant virus based on sample release agent treatment;

[0044] Fig.12 It is the quantitative result of the template copy number of rVSV recombinant virus treated with sample release agent;

[0045] Fig.13 , 14 , 15 are the sensitivity detection results of RT-RPA-CRISPR / Cas12a based on the treatment of rVSV recombinant virus with sample release agent;

[0046] Fig.16 , 17 , 18 are the specific detection results of RT-RPA-CRISPR / Cas12a based on rVSV-infected mice. DETAILED DESCRIPTION

[0047] The technical solution of the present invention is further described below through specific embodiments in conjunction with the accompanying drawings.

[0048] In the following examples of the present invention, LB liquid culture medium and solid LB culture medium were prepared by the laboratory, and the ultra-micro spectrophotometer was purchased from NanoDrop one of Thermo Fisher Scientific.

[0049] Example 1: Acquisition of RT-RPA primers and crRNA sequences

[0050] The full genes of Congo fever virus, Rift Valley fever virus, Nipah virus and Hendra virus that have been published were searched on NCBI GeneBank, and the full genes were sequenced and analyzed by MEGA7.0 software after downloading. Relatively conservative target genes (N, F, G) were screened out by analyzing the results and consulting the literature. Then, MEGA7.0 was used to perform sequence alignment on the target genes to find the core conservative regions. The N genes of Congo fever virus, Rift Valley fever virus, Nipah virus and Hendra virus were selected respectively, and the sequence alignment of the target genes was performed by MEGA7.0 to find the core conservative regions.

[0051] According to the property that Cas12a recognizes PAM as TTTN (N is an arbitrary base), detection targets for Congo fever virus, Rift Valley fever virus, Nipah virus and Hendra virus are designed in the conserved sequence, and the specificity of the detection target is verified using BLAST. The corresponding crRNA target sequence is further designed. RT-RPA primers are designed and screened for both ends of the crRNA of the N gene of Congo fever virus, Rift Valley fever virus, Nipah virus and Hendra virus. The RT-RPA primer sequences and corresponding crRNA sequences are shown in Tables 1-4, respectively.

[0052] Table 1 Congo fever virus RT-RPA primer sequences and crRNA sequences

[0053]

[0054] Table 2 Rift Valley fever virus RT-RPA primer sequences and crRNA sequences

[0055]

[0056] Table 3 Nipah virus RT-RPA primer sequences and crRNA sequences

[0057]

[0058] Table 4 Hendra virus RT-RPA primer sequence and crRNA sequence

[0059]

[0060] Example 2: Identification of viral gene recombinant plasmid

[0061] The primers and target gene sequences selected in Example 1 were respectively delivered to BGI for synthesis to construct recombinant plasmids.

[0062] Transformation, amplification and extraction of recombinant plasmids:

[0063] 1. Transformation of recombinant plasmid

[0064] Take 2μL of recombinant plasmid and add it to a 50μL DH5α competent cell EP tube, and gently blow to mix. Place on ice for 30 minutes, heat shock at 42℃ for 45s, and place on ice for 2-3 minutes. Add 900μL of LB liquid culture medium without resistance, place it in the EP tube, and shake the bacteria on a constant temperature shaker at 37℃ and 220r / min for 1h. Take out the shaking EP tube, centrifuge at 5000r / min for 5min, discard the supernatant, resuspend the bacteria with the remaining culture medium, and use a sterile coating rod to evenly spread the bacteria on the solid LB culture medium plate with the corresponding resistance. Incubate inverted at 37℃ incubator for 12h.

[0065] 2. Amplification of recombinant plasmid

[0066] A single colony on the solid LB medium plate was picked and inoculated into 5 mL of liquid LB medium, and cultured on a constant temperature shaker at 37°C and 220 r / min for 12 h.

[0067] 3. Extraction of recombinant plasmid

[0068] Transfer the bacterial solution cultured for 12 hours in step 2 to a clean 2mL centrifuge tube, centrifuge at 11000rpm for 1min at room temperature, and discard the waste liquid. Use the Omega Plasmid Extraction Type I Kit according to the operating instructions, add 250μL SolutionⅠ, vortex and shake, and mix thoroughly until there is no obvious bacterial precipitation in the tube. Add 250μL SolutionⅡ, mix gently, and let stand for 2-3min. Add 350μL SolutionⅢ, mix gently (milky white flocculent material appears), and centrifuge at 12000rpm for 10min. Gently transfer the supernatant to the adsorption column provided with the kit, centrifuge at 12000rpm for 1min, and discard the waste liquid. Add about 500μL HBCBuffer to the same adsorption column, centrifuge at 12000rpm for 1min, and discard the waste liquid. Add about 700μL DNA Wash Buffer to the same adsorption column, centrifuge at 12000rpm for 1min, and discard the waste liquid. Repeat adding 700μL DNA Wash Buffer, centrifuge at 12000rpm for 1min, and discard the waste liquid. Incubate the adsorption column at 12000rpm for 2min. Leave it uncapped to evaporate the ethanol. Transfer the adsorption column to a new 1.5mL centrifuge tube. Add 35μL sterile ddH2O (deionized water) to the center of the column membrane, cover it and let it stand for 1min, centrifuge at 12000rpm for 1min, and store it at 4℃. Use an ultra-micro spectrophotometer to measure the concentration of the extracted plasmid.

[0069] (1) Recombinant plasmid digestion and identification

[0070] The extracted four viral N gene plasmids (CCHFV-N gene plasmid, RVFV-N gene plasmid, NiV-N gene plasmid and HeV-N gene plasmid) were further double-digested with restriction endonucleases EcoRⅠ and XhoⅠ, and the digestion products were subjected to agarose gel electrophoresis. The bottom band was the viral N gene fragment, and the fragment size was consistent with expectations. Among them, the CCHFV-N gene fragment was digested into two fragments of 330bp and 1157bp, the RVFV-N gene fragment was digested into two fragments of 93bp and 652bp, the NiV-N gene fragment was digested into three fragments of 410bp, 453bp and 745bp, and the HeV-N gene fragment was digested into two fragments of 684bp and 923bp; the results are as follows Figure 1 shown.

[0071] (2) Identification of recombinant plasmid protein expression

[0072] The synthesized CCHFV-N gene plasmid, RVFV-N gene plasmid, NiV-N gene plasmid and HeV-N gene plasmid were transfected into 293T cells respectively. The culture conditions were DMEM+10% FBS+1% P / S, the culture environment was 5% carbon dioxide (CO2), and 37°C. After 18-36 hours, the cell protein samples were collected. Protein immunoblotting experiments were used to verify the expression of CCHFV-N protein, RVFV-N protein, NiV-N protein and HeV-N protein. The above proteins were expressed correctly. The results are as follows Figure 2 shown.

[0073] Example 3: Preparation of CCHFV-N gene recombinant plasmid standard

[0074] According to the recombinant plasmid size and plasmid concentration, the copy number of the plasmid in the plasmid stock solution was calculated according to the following formula:

[0075]

[0076] Y represents the copy number, and X represents the measured plasmid concentration in ng / μL.

[0077] The calculation results are as follows:

[0078] The CCHFV-N plasmid size was 6843 bp and the concentration was 504.7 ng / μL. The calculated copy number was 6.727×10 10 copies / μL; the RVFV-N plasmid size is 6132bp, the concentration is 647.1ng / μL, and the copy number is calculated to be 9.625×10 10 copies / μL; the NiV-N plasmid size is 6993bp, the concentration is 542.6ng / μL, and the copy number is calculated to be 7.077×10 10 copies / μL; the HeV-N plasmid size is 6993bp, the concentration is 711.2ng / μL, and the copy number is calculated to be 9.276×10 10 copies / μL.

[0079] Use TE Buffer to dilute the target gene recombinant plasmid stock solutions of the four viruses to a concentration of 1×10 0 , 1×10 1 , 1×10 2 , 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×1010 The standard samples were divided into 1000 copies / μL and stored in a -80℃ refrigerator for future use.

[0080] Example 4: Validation of crRNA for four virus detections

[0081] The crRNAs of the four designed viruses were verified to determine the applicability of the crRNAs. The crRNAs designed and synthesized for the four viral N genes and the corresponding standard plasmids were used as target templates to perform CRISPR / Cas12a cleavage reactions. When the viral N gene standard plasmids and the corresponding crRNAs and Cas12a proteins were successfully combined into a complex and the trans-cleavage activity was activated, the probes could be cut, and the fluorescence signals were read by a real-time fluorescence PCR instrument to determine the feasibility of the designed crRNA for detecting alternative viruses. At the same time, green fluorescence can be observed under ultraviolet light. The specific reaction system is shown in Table 5.

[0082] Table 5 CRISPR / Cas12a trans-cleavage system

[0083]

[0084]

[0085] The lysis buffer was synthesized by Addygene. The operation steps and program settings are as follows:

[0086] In the clean bench, add each component to the eight-tube according to the system in Table 5, shake and mix thoroughly, and operate on ice and avoid light. Set the reaction program in the ABI 7500 fluorescent quantitative PCR instrument: preheat at 25℃ for 1min, 1 cycle; react at 37℃ for 30s, 60 cycles, and collect the fluorescence signal of the FAM fluorescence channel once every 30s in each cycle. After the reaction, save and analyze the results. After the reaction, place the sample in a dark room and irradiate it with UV light. The results are as follows: Figure 3 , 4 , 5. Only the detected virus showed green fluorescence under UV light, while NC had no fluorescence; and compared with NC, its fluorescence value increased significantly with the cycle.

[0087] Example 5: Screening of primers for four viral RT-RPA

[0088] 1. Obtaining RT-RPA template

[0089] After 48 hours of infection of 293T cells with pseudoviruses of lentiviral vectors carrying four viral conserved N genes preserved in the applicant's laboratory (rLentivirus-CCHFV-N, rLentivirus-RVFV-N, rLentivirus-NiV-N and rLentivirus-HeV-N), total RNA was extracted using the Trizol method and used as a template for experiments.

[0090] 2. RT-RPA primer screening

[0091] Among the primers corresponding to the four viruses, a reverse primer was first randomly selected, and all forward primers were used to amplify the template with the reverse primer. The best forward primer was screened according to the agarose gel electrophoresis results. Then, all reverse primers were used to amplify with the forward primer, and the best reverse primer was screened according to the agarose gel electrophoresis results, and the best primer combination for RT-RPA was finally determined. The RT-RPA kit was purchased from Easy Bio and Ampo Future, and the RT-RPA reaction system is shown in Table 6.

[0092] Table 6 RT-RPA reaction system

[0093]

[0094]

[0095] Among them, rehydration buffer and Starter were purchased from Anpu Future Biotechnology Co., Ltd. The RT-RPA reaction operation steps and program settings are as follows:

[0096] In the clean bench, add each component to the eight tubes containing lyophilized powder according to the system in Table 4 on ice, shake and mix thoroughly, centrifuge briefly, heat in a 39°C water bath for 30 minutes, and perform RT-RPA reaction. However, it should be noted that when configuring the RT-RPA reaction system, Starter must be added last, because Starter will immediately start the RT-RPA reaction. In addition, the dry powder in the eight tubes must be fully dissolved and mixed thoroughly with each component.

[0097] The RT-RPA amplification product needs to be extracted and purified to obtain clear and intuitive results under the gel system. Use DNA extraction reagent (Tris saturated phenol: chloroform: isoamyl alcohol = 25:24:1) to extract DNA from the amplification product. The steps are as follows:

[0098] (1) The extraction reagent and amplification product were mixed thoroughly in a ratio of 1:1 and centrifuged at 4°C and 12,000 rpm for 4 min.

[0099] (2) After centrifugation, the liquid surface is divided into three layers: the upper layer is DNA, the middle layer is protein, and the lower layer is organic matter. Carefully extract the DNA in the upper layer and transfer it to a new enzyme-free centrifuge tube to obtain the product DNA. After obtaining the extracted DNA, add the loading buffer and perform agarose gel electrophoresis to observe the results and determine the best primer combination for RT-RPA. The results are as follows: Figure 6 , 7 As shown. The reverse primer selected for NiV-N is NVN-FA-RB, and the forward primers are NVN-RB-FD and NVN-RB-FF; the reverse primer selected for HeV-N is HVN-FA-RF, and the forward primer is HVN-RF-FE; the reverse primer selected for RVFV-N is RVN-FA-RD, and the forward primer is RVN-RD-F; the reverse primer selected for CCHFV-N is CVN-FA-RF, and the forward primer is CVN-RB-FD.

[0100] Example 6: Optimization of RT-RPA-CRISPR / Cas12a detection system for four viruses

[0101] 1. Optimization of RT-RPA reaction temperature

[0102] Since the optimal reaction temperature of RT-RPA is around 37°C, the reaction temperature range is set to 33-41°C, and the reaction temperatures are set to 33°C, 35°C, 37°C, 39°C, and 41°C respectively. According to the above reaction system and reaction procedure, different reaction temperatures are used for reaction. Finally, nucleic acid electrophoresis and CRISPR / Cas12a are used to detect the RT-RPA reaction products to screen the optimal reaction temperature of RT-RPA. After the reaction is completed, a fluorescence curve is drawn. The results show that the optimal reaction temperature of RT-RPA for the four viruses is 39°C. Figure 8 shown.

[0103] 2. CRISPR / Cas12a reaction temperature optimization

[0104] Since the optimal reaction temperature of CRISPR / Cas12a is also around 37°C, the reaction temperature range is set to 33-41°C, and the reaction temperatures are set to 33°C, 35°C, 37°C, 39°C, and 41°C respectively. According to the above reaction system and reaction procedure, different reaction temperatures are used for reaction, and fluorescence curves are drawn after the reaction. The results show that the optimal reaction temperature of CRISPR / Cas12a for the four viruses is 39°C. Fig. 9 shown.

[0105] Example 7: Performance evaluation of four virus RT-RPA-CRISPR / Cas12a detection systems

[0106] (1) The sample release agent (purchased from Xinda Gene) was used to quickly extract the recombinant viral nucleic acids of rVSV-CCHFV-N, rVSV-RVFV-N, rVSV-NiV-N, and rVSV-HeV-N, and perform RT-RPA-CRISPR / Cas12a specific experiments.

[0107] Since the NiV, HeV, RVFV, and CCHFV involved are all major zoonoses, these four pathogens need to be studied with live viruses in laboratories at level 3 or above. Therefore, the applicant did not conduct live virus research, but instead used the recombinant viruses rVSV-CCHFV-N, rVSV-RVFV-N, rVSV-NiV-N, rVSV-HeV-N and other viruses including avian influenza virus (AIV), Newcastle disease virus (NDV), vesicular stomatitis virus (VSV), lentivirus, Sendai virus (SeV), herpes simplex virus (HSV) and other viruses stored in the applicant's laboratory, extracted nucleic acids from the virus solution using a sample releaser, and conducted RT-RPA-CRISPR / Cas12a specific experiments. The specific steps are as follows: In a biological safety cabinet, take 500 μL of sample release agent and 500 μL of virus stock solution and add them to a 1.5 mL EP tube. After mixing thoroughly, let it stand for 5 minutes.

[0108] The remaining viruses were treated in the same way to obtain nucleic acid templates, which were used as templates and added to the RT-RPA reaction tubes. The reaction system was prepared as described above, the centrifuge tubes were closed, and placed in a 39°C water bath for reaction for 30 minutes. After the amplification was completed, centrifuged instantly. A mixture was prepared according to the CRISPR / Cas12a reaction system, mixed with the RT-RPA amplification product and the cutting reaction was initiated. The centrifuge tubes were placed in the ABI 7500 fluorescence quantitative PCR instrument, and the reaction program was set: preheating at 25°C for 1 minute, 1 cycle; reacting at 37°C for 30 seconds, 60 cycles, and collecting the fluorescence signal of the FAM fluorescence channel once every 30 seconds of each cycle. After the reaction was completed, the results were saved and analyzed. The samples after the reaction were placed in a dark room, irradiated with UV light, observed the results, and photographed for preservation. The results showed that in each system, only the recombinant viruses of the VSV vectors carrying the conserved N genes of the four viruses were detected as positive, showing green fluorescence, such as Fig.10 , 11 shown.

[0109] (2) Use sample release agents to quickly extract rVSV-CCHFV-N, rVSV-RVFV-N, rVSV-NiV-N, and rVSV-HeV-N recombinant viral nucleic acids, and perform RT-RPA-CRISPR / Cas12a sensitivity experiments. Use the rVSV-CCHFV-N, rVSV-RVFV-N, rVSV-NiV-N, and rVSV-HeV-N recombinant viral nucleic acid samples obtained in the above steps as templates. Use the Taqman probe qPCR detection method for the conserved N genes of the four viruses established in the applicant's laboratory in the early stage to quantitatively detect the obtained templates, and calculate the copy number of the template using the standard curve. Then use TE Buffer to dilute the template 10 times to a final concentration of 10 4 -10 0 copies / μL of template.

[0110] The Taqman probe qPCR detection method is as follows:

[0111] Under the optimal Taqman probe qPCR reaction conditions for the four viruses, single-plex Taqman qPCR detection was performed using Premix Ex TaqTM (ProbeqPCR). Three replicate wells were set for the viral target gene RNA template, and three negative controls were set. The reaction systems were prepared as shown in Tables 7-10:

[0112] Table 7 Congo fever virus Taqman qPCR reaction system

[0113]

[0114] Table 8 Rift Valley fever virus Taqman qPCR reaction system

[0115]

[0116] Table 9 Nipah virus Taqman qPCR reaction system

[0117]

[0118]

[0119] Table 10 Hendra virus Taqman qPCR reaction system

[0120]

[0121] The mixed solutions of the above reaction systems were added to 96-well plates, sealed, and reacted on the LightCycler 480 System. The Taqman qPCR reaction conditions were: pre-denaturation: 95°C, 30s, 1 cycle; PCR: 95°C, 5s, 62°C, 30s for a total of 45 cycles, and the fluorescence signal was detected once after each cycle; final extension: 50°C, 30s, 1 cycle. After the reaction, the template copy number was calculated based on the Ct value and the standard curve, and dilution and subsequent experiments were performed. The results are shown in Fig.12 shown.

[0122] Add the template to the RT-RPA reaction tube, configure the reaction system according to the configuration, close the centrifuge tube, and place it in a 39°C water bath for 30 minutes. After the amplification is completed, centrifuge instantly. Configure the mixture according to the CRISPR / Cas12a reaction system, mix it with the RT-RPA amplification product and start the cutting reaction. Place the centrifuge tube in the ABI 7500 fluorescent quantitative PCR instrument, and set the reaction program: preheat at 25°C for 1 minute, 1 cycle; react at 37°C for 30 seconds, 60 cycles, and collect the fluorescence signal of the FAM fluorescence channel once every 30 seconds in each cycle. After the reaction is completed, save and analyze the results. Place the sample after the reaction in a dark room, irradiate with UV light, observe the results, and take pictures for preservation. The results show that the minimum dilution that can be detected for rVSV-CCHFV-N, rVSV-RVFV-N, rVSV-NiV-N and rVSV-HeV-N is 1 copies / μL, which has high sensitivity, such as Fig.13 , 14 , as shown in 15.

[0123] Example 8: Application of four virus RT-RPA-CRISPR / Cas12a detection systems

[0124] C57BL / 6 mice were infected with rVSV-CCHFV-N, rVSV-RVFV-N, rVSV-NiV-N and rVSV-HeV-N respectively. The organs of infected mice were collected to simulate clinical samples. RNA from infected mouse tissues was further extracted, and RT-RPA-CRISPR / Cas12a was used to detect RNA from rVSV recombinant virus infected mouse tissues. Finally, the fluorescence signal was collected using an ABI 7500 fluorescence quantitative PCR instrument, and the results were saved and analyzed after the reaction. The samples after the reaction were placed in a dark room, irradiated with UV light, and the results were observed and photographed for preservation. The results are shown in the figure. Fig.16 , 17 , 18. The RNA of the organs and tissues of the virus-infected mice showed green fluorescence under UV light, while the PBS control group and the NC group showed no fluorescence.

Claims

1. A rapid detection system for virulent viral nucleic acid based on RT-RPA and CRISPR / Cas12a, characterized in that: The virulent virus is Congo fever virus, Rift Valley fever virus, Nipah virus or Hendra virus; the detection system includes an RT-RPA isothermal amplification system and a CRISPR / Cas12a trans-cutting detection system; The RT-RPA isothermal amplification system includes RT-RPA forward primers and reverse primers designed based on viral nucleic acid; Wherein, the nucleotide sequence of the forward primer of Congo fever virus RT-RPA is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2; The nucleotide sequence of the forward primer of Rift Valley fever virus RT-RPA is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.4; The nucleotide sequence of the Nipah virus RT-RPA forward primer is shown in SEQ ID NO.5, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.6; The nucleotide sequence of the Hendra virus RT-RPA forward primer is shown in SEQ ID NO.7, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.8; The CRISPR / Cas12a trans-cleavage detection system includes a crRNA sequence designed based on viral nucleic acid; Wherein, the nucleotide sequence of Congo fever virus crRNA is as shown in SEQ ID NO.9; The nucleotide sequence of Rift Valley fever virus crRNA is as shown in SEQ ID NO.10; The nucleotide sequence of Nipah virus crRNA is as shown in SEQ ID NO.11; The Hendra virus crRNA nucleotide sequence is as shown in SEQ ID NO.

12.

2. A rapid detection system for virulent viral nucleic acid based on RT-RPA and CRISPR / Cas12a as claimed in claim 1, characterized in that: The CRISPR / Cas12a trans-cleavage detection system contains 2 μL of crRNA (500 nM) and also includes: Lysis buffer (10X) 3 μL; LbCas12a nuclease (1 μM) 1 μL; ssDNA fluorescent reporter gene (2 μM) 6 μL; DNA template 2 μL; DEPC-H2O 16 μL.

3. A rapid detection system for virulent viral nucleic acids based on RT-RPA and CRISPR / Cas12a according to claim 2, characterized in that: The RT-RPA isothermal amplification system contains 1 μL of a forward primer (10 μM), 1 μL of a reverse primer (10 μM), and also includes: Rehydration buffer (2X) 10μL; RNA template 2μL; ddH2O 4μL; Starter (10X) 2μL.

4. A rapid detection kit for virulent viral nucleic acid based on RT-RPA and CRISPR / Cas12a, characterized in that: The kit comprises the RT-RPA isothermal amplification system and the CRISPR / Cas12a trans-cleavage detection system as described in any one of claims 1 to 3.

5. A method for using a rapid detection kit for virulent viral nucleic acid based on RT-RPA and CRISPR / Cas12a as claimed in claim 4, characterized in that, The following steps are involved: S1: Add the RNA of the sample to be tested into the RT-RPA amplification system for RT-RPA isothermal amplification to obtain the RT-RPA amplification product; S2: Add the RT-RPA amplification product to the CRISPR / Cas12a detection system for reaction; S3: Real-time detection of fluorescence changes of the CRISPR / Cas12a detection system in S2 and drawing of fluorescence curves; In the drawn fluorescence curve, the test result in which the fluorescence value increases with time is judged as positive, and the result in which the fluorescence value is a horizontal line with time is considered as negative.

6. A method for using a rapid detection kit for virulent viral nucleic acid based on RT-RPA and CRISPR / Cas12a according to claim 5, characterized in that: In step S1, the RT-RPA isothermal amplification conditions are: 39° C. water bath reaction for 30 min.

7. According to the method for using a rapid detection kit for virulent viral nucleic acid based on RT-RPA and CRISPR / Cas12a according to claim 6, in the step S2, the reaction conditions are: preheating at 25°C for 1 min, 1 cycle; reacting at 37°C for 30 s, 60 cycles, and collecting the fluorescence signal of the FAM fluorescence channel once each cycle.

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