Method for rapidly detecting foot and mouth disease virus based on CRISPR / Cas12a system and RPA amplification technology

Abstract

The invention relates to the technical field of molecular diagnosis and virology detection, and discloses a method for rapidly detecting foot-and-mouth disease viruses based on a CRISPR / Cas12a system and an RPA amplification technology, which breaks through the dependence limitation of a traditional detection method on a precise instrument by integrating a dual technical path of RPA isothermal amplification and CRISPR / Cas12a fluorescence detection, and improves the detection accuracy of the foot-and-mouth disease viruses. The whole detection process can be completed under the basic equipment condition, the feasibility of on-site rapid screening is improved, meanwhile, specific recognition is conducted on target genes through the trans-cleavage characteristic of Cas12a, the misjudgment risk caused by cross reaction between serotypes is avoided, the accuracy and reliability of the detection result are guaranteed, and the detection efficiency is improved. By optimizing a Cas12a-crRNA compound pre-incubation mechanism and reaction system dynamic balance control, the interference influence of ambient temperature fluctuation on enzyme activity is overcome, so that the detection system can still maintain stable cutting efficiency under complex field conditions.

Description

Technical Field

[0001] The present invention relates to the field of molecular diagnosis and virological detection technology, and specifically to a method for rapid detection of foot-and-mouth disease virus based on the CRISPR / Cas12a system and RPA amplification technology. Background Art

[0002] Foot-and-mouth disease is an acute, febrile, highly contagious disease of even-toed ungulates caused by the foot-and-mouth disease virus. It mainly attacks even-toed ungulates and occasionally occurs in humans and other animals. Its clinical characteristics are blisters on the oral mucosa, hooves and udder skin.

[0003] In the current practice of foot-and-mouth disease virus detection, conventional RT-PCR technology seriously restricts the ability to quickly respond to epidemics at the scene due to its core limitations. This technology involves four independent operations: RNA extraction, reverse transcription, PCR amplification, and electrophoresis analysis. The entire process is time-consuming and relies on high-precision qPCR equipment, resulting in insufficient penetration of grassroots quarantine stations.

[0004] Therefore, a method for rapid detection of foot-and-mouth disease virus based on CRISPR / Cas12a system and RPA amplification technology is proposed to solve the above problems. Summary of the Invention

[0005] (1) Technical problems solved

[0006] In response to the shortcomings of the existing technology, the present invention provides a method for rapid detection of foot-and-mouth disease virus based on the CRISPR / Cas12a system and RPA amplification technology, which solves the problems raised in the above background technology.

[0007] (2) Technical solution

[0008] To achieve the above objectives, the present invention provides the following technical solutions: a method for rapid detection of foot-and-mouth disease virus based on the CRISPR / Cas12a system and RPA amplification technology, comprising the following steps:

[0009] Step 1: Extract viral RNA. Use an RNA extraction kit containing lysis buffer RZ to treat the sample, and obtain RNA with a purity of ≥1.8 by chloroform layer separation, centrifugation, ethanol precipitation, and adsorption column purification.

[0010] Step 2: cDNA synthesis: RNA was treated with a reverse transcriptase system containing 1.5 μL RNA template, 1 μL random primers (0.1 μg / μL), 1 μL 10 mM dNTPs, 4 μL 5× RT Buffer, 0.5 μL ribonuclease inhibitor, 1 μL reverse transcriptase, and RNase-free water to 20 μL. The reaction was pre-incubated at 25°C for 5 minutes and then at 42°C for 30 minutes.

[0011] Step 3: RPA amplification: Recombinase polymerase amplification was performed using a primer pair targeting the 3D pol gene of foot-and-mouth disease virus. The upstream primer sequence was 5'-AAAAGCGACAAAGGTTTTGTTCTTGGTCACTCCAT-3';

[0012] The downstream primer sequence was 5'-GTTCACCCAACGCAGGTAAAGTGATCTGTAGCTTG-3'. The reaction system contained 29.5 μL Rehydration Buffer, 11.2 μL ddH2O, 1.2 μL upstream primer, 1.2 μL downstream primer, 1 μL cDNA template, and 1.25 μL MgOAc. The reaction was incubated in a 39°C ± 1°C water bath for 20 min.

[0013] Step 4: CRISPR / Cas12a fluorescence detection: Mix Cas12a protein, specific crRNA, NEBuffer r2.1, ssDNA fluorescent reporter molecule and RPA amplification product of step 3, where:

[0014] The crRNA sequence is 5'-UAAUUUCUACUAAGUGUAGAUCUCCUGUAUGGUCCCACGCGUG-3', the ssDNA reporter molecule is 5'-FAM-TTATT-3'-BHQ1, and the reaction system consists of: 21.5 μL ddH2O, 1 μL Cas12a: 30-100 nmol / L, 1 μL crRNA: 50-100 nmol / L, 3 μL NEBuffer r2.1, 0.5 μL ssDNA: 250-450 nmol / L, 3 μL RPA product, first react at 25°C for 10 minutes to form a Cas12a-crRNA complex, and then incubate at 37°C ± 2°C for 20 minutes;

[0015] Step 5: Result determination: real-time fluorescence quantitative PCR instrument FAM channel detection, fluorescence intensity ≥ 1000RFU is considered positive.

[0016] Preferably, the step 1 specifically includes:

[0017] Lysis: 1 mL of lysis buffer RZ was treated at room temperature for 5 minutes;

[0018] Layer separation: add 200 μL chloroform, shake for 15 seconds, and let stand for 3 minutes;

[0019] Centrifugation: 12,000 rpm at 4°C for 10 minutes to obtain the aqueous phase;

[0020] Precipitation: add 0.5 times the volume of anhydrous ethanol and mix well;

[0021] Purification: centrifuge the adsorption column for 30 seconds and wash twice with deproteinization solution;

[0022] Elution: Elute RNA with 50 μL RNase-free water.

[0023] Preferably, the RPA amplification satisfies the following parameter ranges:

[0024] Primer concentration: 8-12 μmol / L;

[0025] MgOAc addition amount: 1.0-1.5 μL;

[0026] Temperature fluctuation: ±1℃;

[0027] Time deviation: ±5 minutes.

[0028] Preferably, the two ends of the ssDNA reporter molecule are labeled with FAM and BHQ1 groups, respectively, with the concentration optimization range of 250-450 nmol / L and the optimal concentration of 350 nmol / L. When Cas12a activates cleavage, FAM and BHQ1 separate and release fluorescence.

[0029] Preferably, the crRNA sequence is designed as follows:

[0030] 5'-UAAUUUCUACUAAGUGUAGAUCUCCUGUAUGGUCCCACGCGUG-3', which contains:

[0031] I, palindromic backbone 5′-UAAUUUCUACUAAGUGUAGAU-3′;

[0032] II. Target complementary region, corresponding to the PAM flanking sequence 5'-CACGCCGUGGGACCAUACAGGAG-3.

[0033] Preferably, a three-level gradient is used:

[0034] Cas12a gradient: 15 nmol / L, 30 nmol / L, 50 nmol / L, 100 nmol / L;

[0035] crRNA gradient: 30 nmol / L, 50 nmol / L, 100 nmol / L;

[0036] The fluorescence intensity of the combination of 50nmol / L Cas12a+50nmol / L crRNA reached a peak of 3077RFU.

[0037] Preferably, the reaction time of step 4 is optimized by chessboard titration, comprising:

[0038] Complex formation time: 5 / 10 / 15 / 20 / 25 / 30 min gradient;

[0039] Activation reaction time: 5 / 10 / 15 / 20 / 25 / 30 minutes gradient;

[0040] The optimal condition was that the complex was formed for 10 minutes and then reacted for 20 minutes, and the fluorescence intensity reached 3463RFU.

[0041] Preferably, the sensitivity verification shows that the detection limit is 10 2 copies / μL, standard sample gradient dilution, 10 2 The fluorescence signal of the samples with copies / μL was 3 times the standard deviation higher than the background value.

[0042] Preferably, in the specificity verification, the fluorescence intensity of samples of transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine circovirus type 2, porcine circovirus type 3, swine vesicular disease virus, vesicular stomatitis virus and porcine Seneca virus is less than 500RFU, while that of foot-and-mouth disease virus sample is greater than 3000RFU.

[0043] Preferably, the whole process time control is:

[0044] RNA extraction ≤ 15 min;

[0045] Reverse transcription ≤ 30 min;

[0046] RPA amplification ≤ 20 min;

[0047] CRISPR assay ≤ 30 minutes;

[0048] And only a water bath, a centrifuge and a portable fluorescence instrument are needed to complete the test.

[0049] (3) Beneficial effects

[0050] Compared with the existing technology, the present invention provides a method for rapid detection of foot-and-mouth disease virus based on the CRISPR / Cas12a system and RPA amplification technology, which has the following beneficial effects:

[0051] 1. In the present invention, in the process of foot-and-mouth disease virus detection, by integrating the dual technical paths of RPA isothermal amplification and CRISPR / Cas12a fluorescence detection, the dependence of traditional detection methods on precision instruments is broken through, so that the entire detection process can be completed under basic equipment conditions, improving the feasibility of on-site rapid screening, and at the same time utilizing the trans-cleavage characteristics of Cas12a to specifically identify the target gene, avoiding the risk of misjudgment caused by cross-reactions between serotypes, and ensuring the accuracy and reliability of the test results.

[0052] 2. In the present invention, in the viral nucleic acid amplification link, by optimizing the Cas12a-crRNA complex pre-incubation mechanism and the dynamic balance control of the reaction system, the interference of ambient temperature fluctuations on enzyme activity is overcome, so that the detection system can still maintain a stable cutting efficiency under complex field conditions. At the same time, a dual-threshold verification logic is established in the fluorescence signal interpretation stage, and the review procedure is automatically triggered when a nonspecific background signal is generated, thereby reducing the probability of false negative results and ensuring the timeliness and accuracy of the initial screening of the epidemic.

[0053] 3. In the present invention, in the construction of the entire detection process, the operational sequence of the four steps of RNA extraction, reverse transcription, RPA amplification and CRISPR detection is modularly integrated to realize the integrated operation paradigm of "sample in - result out", so that the operator does not need to transfer the reaction system in stages, thereby avoiding the risk of aerosol contamination from the root. Based on the adaptive calibration mechanism of fluorescence signal intensity, gradient response recognition is realized for low viral load samples, thereby improving the detection system's ability to capture latently infected samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is a graph showing the results of ssDNA fluorescent probe concentration optimization in Example 2 of the present invention;

[0055] Figure 2 This is a sensitivity result diagram in Example 5 of the present invention;

[0056] Figure 3 This is a diagram of the CRISPR / Cas12a specificity results in Example 6 of the present invention. DETAILED DESCRIPTION

[0057] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0058] Example 1:

[0059] The method comprises the following steps:

[0060] Step 1: Extract viral RNA. Use an RNA extraction kit containing lysis buffer RZ to treat the sample, and obtain RNA with a purity of ≥1.8 by chloroform layer separation, centrifugation, ethanol precipitation, and adsorption column purification.

[0061] Step 2: cDNA synthesis: RNA was treated with a reverse transcriptase system containing 1.5 μL RNA template, 1 μL random primers (0.1 μg / μL), 1 μL 10 mM dNTPs, 4 μL 5× RT Buffer, 0.5 μL ribonuclease inhibitor, 1 μL reverse transcriptase, and RNase-free water to 20 μL. The reaction was pre-incubated at 25°C for 5 minutes and then at 42°C for 30 minutes.

[0062] Step 3: RPA amplification: Recombinase polymerase amplification was performed using a primer pair targeting the 3D pol gene of foot-and-mouth disease virus. The upstream primer sequence was 5'-AAAAGCGACAAAGGTTTTGTTCTTGGTCACTCCAT-3';

[0063] The downstream primer sequence was 5'-GTTCACCCAACGCAGGTAAAGTGATCTGTAGCTTG-3'. The reaction system contained 29.5 μL Rehydration Buffer, 11.2 μL ddH2O, 1.2 μL upstream primer, 1.2 μL downstream primer, 1 μL cDNA template, and 1.25 μL MgOAc. The reaction was incubated in a 39°C ± 1°C water bath for 20 min.

[0064] Step 4: CRISPR / Cas12a fluorescence detection: Mix Cas12a protein, specific crRNA, NEBuffer r2.1, ssDNA fluorescent reporter molecule and RPA amplification product of step 3, where:

[0065] The crRNA sequence is 5'-UAAUUUCUACUAAGUGUAGAUCUCCUGUAUGGUCCCACGCGUG-3', the ssDNA reporter molecule is 5'-FAM-TTATT-3'-BHQ1, and the reaction system consists of: 21.5 μL ddH2O, 1 μL Cas12a: 30-100 nmol / L, 1 μL crRNA: 50-100 nmol / L, 3 μL NEBuffer r2.1, 0.5 μL ssDNA: 250-450 nmol / L, 3 μL RPA product, first react at 25°C for 10 minutes to form a Cas12a-crRNA complex, and then incubate at 37°C ± 2°C for 20 minutes;

[0066] Step 5: Result determination: real-time fluorescence quantitative PCR instrument FAM channel detection, fluorescence intensity ≥ 1000RFU is considered positive.

[0067] 1. Design of RPA primer sequences for foot-and-mouth disease virus strains

[0068] 1. Design of RPA primer sequences for foot-and-mouth disease virus strains

[0069] The Primer-BLAST tool on the NCBI website was used to search and compare the homology of this fragment in foot-and-mouth disease virus, and the amplification target was determined to be FMDV 3Dpol as the target gene sequence. Based on the amplification target sequence, the RPA primer pair was obtained using Primer Premier5 software. The specific primer sequences are shown in Table 1.

[0070] Table 1 RPA primer sequence information

[0071]

[0072]

[0073] 2. Foot-and-mouth disease virus genome extraction and reverse transcription

[0074] Extraction using total RNA extraction kit:

[0075] (1) Sample processing: Add 1 mL of lysis buffer RZ to each tube and incubate at room temperature for 5 minutes to allow the nucleic acid-protein complex to completely separate.

[0076] (2) Add 200 μL of chloroform, cap the tube, shake vigorously for 15 seconds, and let it stand at room temperature for 3 minutes;

[0077] (3) Centrifuge at 12,000 rpm (~13,400 × g) at 4°C for 10 min, transfer the aqueous phase to a new tube, and proceed to the next step;

[0078] (4) Slowly add 0.5 times the volume of anhydrous ethanol and mix thoroughly. Transfer the resulting solution and precipitate to an adsorption column and centrifuge at 12,000 rpm (~13,400 × g) at 4°C for 30 seconds. Discard the waste liquid in the collection tube.

[0079] (5) Add 500 μL of deproteinized solution containing ethanol to the adsorption column, centrifuge at 12,000 rpm at 4°C for 30 seconds, discard the waste liquid, and place the adsorption column in a collection tube;

[0080] (6) Repeat step 5;

[0081] (7) Place the adsorption column in a 2 mL collection tube and centrifuge at 12,000 rpm for 2 minutes at 4°C to remove any residual liquid.

[0082] (8) Transfer the adsorption column to a new 1.5 mL centrifuge tube, add 50 μL of RNase-Free ddH2O, incubate at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes at 4°C.

[0083] Reverse transcription: Reverse Transcriptase[M-mLV,RNaseH-].

[0084] (1) First-strand cDNA synthesis:

[0085] Table 2 First-strand cDNA synthesis system

[0086]

[0087]

[0088] (3) Mix gently, incubate at 25°C for 5 minutes, and then incubate at 42°C for 30 minutes to obtain the cDNA template.

[0089] 2. RPA Amplification

[0090] Dilute the upstream and downstream primers to 10 μM, respectively, and amplify the target fragment using the TwistAmp™ Basic Kit RPA Amplification Kit. The RPA amplification reaction system is as follows: add 29.5 μL of Rehydration Buffer and 11.2 μL of ddH2O to a reaction tube containing lyophilized enzyme. Centrifuge to completely dissolve the lyophilized enzyme. Then, pipette 20.35 μL of the reaction solution into a new reaction tube, add 1.2 μL of the upstream and downstream primers, 1 μL of the DNA template, centrifuge to mix, add 1.25 μL of MgoAc, and incubate in a water bath at 39°C for 20 minutes.

[0091] 3. Synthesis of single-stranded DNA reporter molecules

[0092] A single-stranded DNA reporter molecule was designed for fluorescence signal detection. This 5-mer random single-stranded DNA molecule, endorsing a FAM and BHQ1 moiety, was synthesized at Qingke Biotechnology. The sequence of the designed and synthesized single-stranded DNA reporter molecule is shown in Table 3.

[0093] Table 3 Single-stranded DNA reporter molecule sequences

[0094] name sequence FQ-Reporter 5′FAM-TTATT-3′BHQ1

[0095] 4. Design and Synthesis of crRNA

[0096] The specific binding and cleavage of most Cas proteins to target dsDNA relies on the crRNA-guided Cas protein's recognition of the PAM in the target sequence, requiring a PAM sequence near the crRNA binding site. The PAM sequence of the LbaCas12a protein is: TTTN, where N is any nucleotide. The specific target nucleic acid sequence is 5'-CACGCCGUGGGACCAUACAGGAG-3'. Based on crRNA design principles, the crRNA reverse complement sequence is added after the palindrome: 5'-UAAUUUCUACUAAGUGUAGAU-3'. The synthetic crRNA sequence is as follows: UAAUUUCUACUAAGUGUAGAUCUCCUGUAUGGUCCCACGGCGUG.

[0097] 5. CRISPR / Cas12a Detection System

[0098] The CRISPR / Cas12a detection system is as follows: Cas12a, specific crRNA, NEBuffer 2.1, and ddH2O are added to a reaction tube, centrifuged and mixed, and then reacted at 25°C for 10 minutes. The purpose is to promote the formation of a complex between Cas12a and specific crRNA, thereby more effectively identifying the target sequence and improving the cleavage efficiency. After the reaction is completed, ssDNA probes and RPA amplification products are added, reacted at 37°C for 20 minutes, and CRISPR fluorescence detection is performed in the FAM channel of a real-time fluorescence quantitative PCR instrument. If a fluorescent signal is collected, it is judged as positive; if no fluorescent signal appears, it is judged as negative. The Cas12a detection system is shown in Table 4.

[0099] Table 4 Cas12a detection system

[0100] Components volume ddH2O 21.5μL Cas12a 1 μL crRNA 1 μL NEBuffer r2.1 3μL ssDNA 0.5μL RPA amplification products 3μL total 30 μL

[0101] 6. CRISPR / Cas12a Detection System Optimization

[0102] 1. Optimization of ssDNA fluorescent probe concentration

[0103] According to the CRISPR detection system, the concentrations of the ssDNA fluorescent probe were set to 450 nmol / L, 400 nmol / L, 350 nmol / L, 300 nmol / L, 250 nmol / L, and 200 nmol / L, respectively. The reaction was carried out at 37°C for 20 minutes, and the fluorescence signal was collected using the FAM channel.

[0104] 2. Optimization of Cas12a and crRNA concentrations

[0105] The optimal probe concentration was used to optimize the concentrations of Cas12a and crRNA. The Cas12a concentration was set to 100 nmol / L, 50 nmol / L, 30 nmol / L, and 15 nmol / L, and the crRNA concentration was set to 100 nmol / L, 50 nmol / L, and 30 nmol / L, and the two were orthogonally tested according to the steps. The fluorescence signal was collected using the FAM channel.

[0106] 3. Response time optimization

[0107] Cas12a, specific crRNA, NEBuffer r2.1, and ddH2O were added to the reaction tube and reacted at 25°C for 30 minutes. A checkerboard titration test was performed, that is, ssDNA probes and RPA amplification products were added at different time periods, mixed, and incubated at 37°C for 30 minutes. Fluorescence signals were collected every 5 minutes, and the strength of the fluorescence signals collected at each time period was compared.

[0108] 4. Sensitivity Verification

[0109] The foot-and-mouth disease virus standard was serially diluted to adjust its concentration to 107, 106, 105, 104, 103, 102, 101, and 100 copies / μL. Total RNA was extracted and reverse transcribed into cDNA according to step 1. RPA amplification was performed according to the amplification system in step 3. The cells were cut according to the optimized CRISPR fluorescence detection system. Each dilution was repeated 3 times. The fluorescence signal was collected using the FAM channel to determine the minimum detection limit of the CRISPR fluorescence method.

[0110] 5. Specificity Verification

[0111] The specificity test was carried out using the genomes of porcine transmissible gastroenteritis virus (GEV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV) and porcine Seneca virus (SVA) as samples. At the same time, a negative control was set up. The optimized CRISPR fluorescence detection system was used for cutting, and the FAM channel was used to collect the fluorescence signal to determine the specificity of the CRISPR fluorescence method.

[0112] Example 2:

[0113] like Figure 1 As shown, the operation was performed according to the CRISPR / Cas12a detection system. The results showed that when the ssDNA probe concentration was greater than 450 nmol / L, the fluorescence value reached a plateau, and when the ssDNA concentration was 450 nmol / L, the detected fluorescence value was the highest. The ssDNA probe concentration of 450 nmol / L was selected as the final reaction concentration of this method.

[0114] Example 3:

[0115] As shown in Table 5, according to the optimized CRISPR / Cas12a detection system, the Cas12a concentration was set to 100nmol / L, 50nmol / L, 30nmol / L, and 15nmol / L, and the crRNA concentration was set to 100nmol / L, 50nmol / L, and 30nmol / L, and the two were subjected to an orthogonal test. The results showed that when the Cas12a concentration was 100nmol / L and the crRNA concentration was 50nmol / L, the detected fluorescence value was up to 3077RFU, so the Cas12a concentration of 100nmol / L and the crRNA concentration of 50nmol / L were used as the final reaction concentrations of the two in the method.

[0116] Table 5. Optimization results of Cas12a and crRNA concentrations

[0117]

[0118]

[0119] Example 4:

[0120] As shown in Table 6, Cas12a, specific crRNA, NEBuffer r2.1, and ddH2O were added to a reaction tube and reacted at 25°C for 30 minutes, and a checkerboard titration test was performed, i.e., ssDNA probes and RPA amplification products were added at different time periods, mixed and incubated at 37°C for 30 minutes, and fluorescence signals were collected every 5 minutes. The results showed that Cas12a, specific crRNA, NEBuffer r2.1, and ddH2O were added to a reaction tube and reacted at 25°C for 10 minutes, ssDNA probes and RPA amplification products were added, mixed and incubated at 37°C for 20 minutes, and the collected fluorescence values ​​were as high as 3463RFU. Therefore, it can be determined that the reaction conditions of this method are: Cas12a, specific crRNA, NEBuffer r2.1, and ddH2O were added to a reaction tube and reacted at 25°C for 10 minutes, then ssDNA probes and RPA amplification products were added, mixed and incubated at 37°C for 20 minutes.

[0121] Table 6. Reaction time optimization results

[0122]

[0123] Example 5:

[0124] Dilute the foot-and-mouth disease virus standard in multiple ratios to adjust its concentration to 10 7 , 10 6 , 10 5, 10 4 , 10 3 , 10 2 , 10 1 , 10 0 Copi es / μL, respectively, extracted total RNA and reverse transcribed into cDNA, and tested using the optimized CRISPR / Cas12a detection system, and each dilution was repeated 3 times. Figure 2 It can be seen that the detection sensitivity of this method can reach 100 copies / μL, which shows that the detection sensitivity of this method is high.

[0125] Example 6:

[0126] The genomes of porcine transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), swine vesicular disease virus (SVDV), vesicular stomatitis virus (VSV), and porcine Senecavirus (SVA) were used as samples. Specificity tests were performed according to the optimized CRISPR / Cas12a detection system, and negative controls were set up. Figure 3 It can be seen that this method has good specificity and can differentiate and diagnose foot-and-mouth disease virus from other viruses.

[0127] It should be noted that, in this document, relational terms such as first and second, etc., are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply the existence of any such actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or device. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or device comprising the element.

[0128] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for rapid detection of foot-and-mouth disease virus based on the CRISPR / Cas12a system and RPA amplification technology, characterized by: The following steps are involved: Step 1: Extract viral RNA. Use an RNA extraction kit containing lysis buffer RZ to treat the sample, and obtain RNA with a purity of ≥1.8 by chloroform layer separation, centrifugation, ethanol precipitation, and adsorption column purification. Step 2: cDNA synthesis: RNA was treated with a reverse transcriptase system containing 1.5 μL RNA template, 1 μL random primers (0.1 μg / μL), 1 μL 10 mM dNTPs, 4 μL 5× RT Buffer, 0.5 μL ribonuclease inhibitor, 1 μL reverse transcriptase, and RNase-free water to 20 μL. The reaction was pre-incubated at 25°C for 5 minutes and then at 42°C for 30 minutes. Step 3: RPA amplification: Recombinase polymerase amplification was performed using a primer pair targeting the 3D pol gene of foot-and-mouth disease virus. The upstream primer sequence was 5'-AAAAGCGACAAAGGTTTTGTTCTTGGTCACTCCAT-3'; The downstream primer sequence was 5'-GTTCACCCAACGCAGGTAAAGTGATCTGTAGCTTG-3'. The reaction system contained 29.5 μL Rehydration Buffer, 11.2 μL ddH2O, 1.2 μL upstream primer, 1.2 μL downstream primer, 1 μL cDNA template, and 1.25 μL MgOAc. The reaction was incubated in a 39°C ± 1°C water bath for 20 min. Step 4: CRISPR / Cas12a fluorescence detection: Mix Cas12a protein, specific crRNA, NEBuffer r2.1, ssDNA fluorescent reporter molecule and RPA amplification product of step 3, where: The crRNA sequence is 5'-UAAUUUCUACUAAGUGUAGAUCUCCUGUAUGGUCCCACGCGUG-3', the ssDNA reporter molecule is 5'-FAM-TTATT-3'-BHQ1, and the reaction system consists of: 21.5 μL ddH2O, 1 μL Cas12a: 30-100 nmol / L, 1 μL crRNA: 50-100 nmol / L, 3 μL NEBuffer r2.1, 0.5 μL ssDNA: 250-450 nmol / L, 3 μL RPA product, first react at 25°C for 10 minutes to form a Cas12a-crRNA complex, and then incubate at 37°C ± 2°C for 20 minutes; Step 5: Result determination: real-time fluorescence quantitative PCR instrument FAM channel detection, fluorescence intensity ≥ 1000RFU is considered positive.

2. A method for rapid detection of foot-and-mouth disease virus based on CRISPR / Cas12a system and RPA amplification technology according to claim 1, characterized in that: The step 1 specifically includes: Lysis: 1 mL of lysis buffer RZ was treated at room temperature for 5 minutes; Layer separation: add 200 μL chloroform, shake for 15 seconds, and let stand for 3 minutes; Centrifugation: 12,000 rpm at 4°C for 10 minutes to obtain the aqueous phase; Precipitation: add 0.5 times the volume of anhydrous ethanol and mix well; Purification: centrifuge the adsorption column for 30 seconds and wash twice with deproteinization solution; Elution: Elute RNA with 50 μL RNase-free water.

3. A method for rapid detection of foot-and-mouth disease virus based on CRISPR / Cas12a system and RPA amplification technology according to claim 1, characterized in that: The RPA amplification meets the following parameter ranges: Primer concentration: 8-12 μmol / L; MgOAc addition amount: 1.0-1.5 μL; Temperature fluctuation: ±1℃; Time deviation: ±5 minutes.

4. A method for rapid detection of foot-and-mouth disease virus based on CRISPR / Cas12a system and RPA amplification technology according to claim 1, characterized in that: The two ends of the ssDNA reporter molecule are labeled with FAM and BHQ1 groups, respectively. The concentration optimization range is 250-450 nmol / L, and the optimal concentration is 350 nmol / L. When Cas12a activates cleavage, FAM and BHQ1 separate and release fluorescence.

5. A method for rapid detection of foot-and-mouth disease virus based on CRISPR / Cas12a system and RPA amplification technology according to claim 1, characterized in that: The crRNA sequence is designed to be 5'-UAAUUUCUACUAAGUGUAGAUCUCCUGUAUGGUCCCACGCGUG-3', which comprises: I, palindromic backbone 5′-UAAUUUCUACUAAGUGUAGAU-3′; II. Target complementary region, corresponding to the PAM flanking sequence 5'-CACGCCGUGGGACCAUACAGGAG-3'.

6. A method for rapid detection of foot-and-mouth disease virus based on CRISPR / Cas12a system and RPA amplification technology according to claim 1, characterized in that: Use a three-level gradient: Cas12a gradient: 15 nmol / L, 30 nmol / L, 50 nmol / L, 100 nmol / L; crRNA gradient: 30 nmol / L, 50 nmol / L, 100 nmol / L; The fluorescence intensity of the combination of 50nmol / L Cas12a+50nmol / L crRNA reached a peak of 3077RFU.

7. A method for rapid detection of foot-and-mouth disease virus based on CRISPR / Cas12a system and RPA amplification technology according to claim 1, characterized in that: Step 4: The reaction time was optimized by chessboard titration, including: Complex formation time: 5 / 10 / 15 / 20 / 25 / 30 min gradient; Activation reaction time: 5 / 10 / 15 / 20 / 25 / 30 minutes gradient; The optimal condition was that the complex was formed for 10 minutes and then reacted for 20 minutes, and the fluorescence intensity reached 3463RFU.

8. The method for rapid detection of foot-and-mouth disease virus based on CRISPR / Cas12a system and RPA amplification technology according to claim 1, characterized in that: Sensitivity verification showed that the detection limit was 10 2 copies / μL, standard sample gradient dilution, 10 2 The fluorescence signal of the samples with copies / μL was 3 times the standard deviation higher than the background value.

9. A method for rapid detection of foot-and-mouth disease virus based on CRISPR / Cas12a system and RPA amplification technology according to claim 1, characterized in that: In the specificity verification, the fluorescence intensity of samples of porcine transmissible gastroenteritis virus, porcine epidemic diarrhea virus, porcine circovirus type 2, porcine circovirus type 3, swine vesicular disease virus, vesicular stomatitis virus and porcine Seneca virus were all less than 500RFU, while that of the foot-and-mouth disease virus sample was greater than 3000RFU.

10. The method for rapid detection of foot-and-mouth disease virus based on CRISPR / Cas12a system and RPA amplification technology according to claim 1, characterized in that: The whole process time control is as follows: RNA extraction ≤ 15 min; Reverse transcription ≤ 30 min; RPA amplification ≤ 20 min; CRISPR assay ≤ 30 minutes; And only a water bath, a centrifuge and a portable fluorescence instrument are needed to complete the test.

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