Capripoxvirus typing detection kit and test strip kit based on array mode and CRISPR / Cas12a

Abstract

The present invention discloses a capripoxvirus typing detection kit and a test strip kit based on an array mode and CRISPR / Cas12a. First, PCR amplification is performed based on the VARV B22R and RPO 30 genes, and the amplified products contain specific regions of LSDV, SPPV and GTPV. The amplified products are then specifically identified by the CRISPR / Cas12a system. A fluorescent array mode is constructed based on two pairs of primers and three CRISPR / Cas12a systems to accurately and specifically identify LSDV, SPPV and GTPV. Under the CRISPR / Cas12a system and PCR amplification mode, the actual detection of LSDV, SPPV and GTPV is as low as 50 copies, 40 copies, and 60 copies, respectively. In addition, the use of CRISPR / Cas12a special test strips combined with the array mode realizes Point-of-care detection of CaPV typing, which is convenient for farmers or enterprises to monitor and identify the virus. Compared with other typing detection methods, the present invention can more accurately and comprehensively identify LSDV, SPPV and GTPV based on the array mode, and has higher sensitivity.

Description

Technical Field

[0001] The present invention belongs to the field of biological virus detection, and in particular relates to a PCR and CRISPR / Cas12a system and an array detection mode capripoxvirus typing detection method. Background Art

[0002] The Capripoxvirus genus (CaPV) within the Chordopoxvirinae family includes sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), which cause sheeppox, goatpox, and lumpy skin disease, respectively, in parts of Africa, the Middle East, and Asia. The viruses are primarily transmitted through direct contact between infected and uninfected animals, indirect contact via aerosols, respiratory droplets, or oral and nasal secretions from acutely infected animals, and mechanical transmission via abrasions or arthropod vectors. Due to their high morbidity and high infectivity, these viruses cause substantial cumulative economic losses and can have devastating impacts on the livelihoods and food security of smallholder farmers, as well as damage to leather and hides, restricting international trade. They are listed as notifiable animal diseases by the World Organization for Animal Health (OIE) and as a category 1 infectious disease in the "List of Quarantine Diseases for Animals Entering the People's Republic of China." However, the epidemiology of Capripox disease is complex, with numerous studies suggesting that it is not strictly host-specific. Analysis of CaPV genome sequences indicates that SPPV, GTPV, and LSDV are genetically distinct and should therefore be defined as three distinct species. However, most isolates of viruses that cause sheeppox and goatpox disease appear to be equally infectious and pathogenic in sheep and goats. Unfortunately, the coexistence of all three forms of the disease in many sub-Saharan countries complicates the identification of the virus causing an outbreak and presents significant challenges in the selection of vaccine control approaches. Therefore, CaPV genotyping is crucial to rapidly and accurately identify the pathogen and facilitate the selection of appropriate control and eradication measures, such as the most appropriate viral vaccine during an outbreak.

[0003] However, serological typing of CaPV is difficult to achieve. Molecular biological methods are more valuable because the antigens of SPPV and GTPV are very similar. Genomic studies have revealed that the genomes of LSDV, SPPV, and GTPV are approximately 150 kb and share at least 147 coding genes, including conserved viral replication and structural genes as well as genes potentially involved in virulence and host range. Sequence alignment revealed that SPPV and GTPV share 96% nucleotide identity over their entire lengths. The SPPV and GTPV genomes are very similar to the genome of lumpy skin disease virus (LSDV), sharing 97% nucleotide identity. Furthermore, all SPPV and GTPV genes are present in LSDV. Notably, nine LSDV genes with potential virulence and host range functions are disrupted in the SPPV and GTPV genomes. The absence of these genes in SPPV and GTPV suggests their important roles in the cattle host range. The SPPV and GTPV genomes contain specific nucleotide differences, indicating that they are phylogenetically distinct. Genomic research found that there are mostly single-base polymorphisms between SPPV and GTPV, which increases the difficulty of identification.

[0004] Extensive sequence alignment revealed that LSDV had inserted 34-36 nucleotides into the gene encoding DNA ligase, VARV B22R (SPPVORF134), and homologous genes, while SPPV and GTPV had no insertions or deletions. Furthermore, there was a 10-nucleotide variation between SPPV field isolates and GTPV. Therefore, this difference in the interval could be exploited to distinguish LSDV. Furthermore, SPPV isolates had a unique 21-nucleotide deletion in the CaPV homolog of the vaccinia virus E4L gene, which encodes the 30 kDa DNA-dependent RNA polymerase subunit, RPO 30, while LSDV and GTPV did not. This deletion was therefore used to develop a classical PCR test based on RPO 30 to distinguish SPPV from GTPV.

[0005] Yana Pestova has recently performed PCR using the LSDV010 ORF as a target gene, employing high-resolution melting curve analysis to analyze LSDV, GTPV, and SPPV. They found that base differences between them cause differences in melting temperatures. Leveraging this property, the method achieved a detection limit as low as 0.1 TCD50 lg / ml. However, the temperature differences are very small, and the method requires expensive equipment and specialized personnel, hindering its widespread application. Separately, Zhixun Zhao used duplex PCR to simultaneously amplify SPPV E10R and RPO132, using agarose gel electrophoresis to analyze the results. The primers designed for E10R amplified the target fragment from both the SPPV and GTPV genomes; however, the primers for the SPPVRPO132 gene amplified only the target gene from the SPPV genome, not the GTPV genome. In other words, SPPV has two distinct target genes, while GTPV only amplifies one. Although PCR combined with agarose gel electrophoresis is the most common method, it is not sensitive enough and cannot detect samples with low concentrations, resulting in false negatives. Therefore, an accurate, sensitive, and rapid method for field-based CaPV typing is needed and desired.

[0006] The CRISPR / Cas12a system, a defense system derived from bacteria and archaea, was discovered in 2012. It possesses strong specificity and signal amplification capabilities. The CRISPR / Cas12a system possesses both cis- and trans-cleavage activities, a key reason for its popularity as a sensor in detection. Specifically, Cas12a utilizes a protospacer-specific motif (PAM) site and CRISPR RNA (crRNA) to complement the target for target recognition. After the target, crRNA, and Cas12a form a ternary complex, Cas12a activates its activity, cleaving the target. Simultaneously, its trans-cleavage activity cleaves surrounding single-stranded DNA. Utilizing the PAM site and crRNA to specifically recognize the target significantly improves specificity and mitigates the problem of false positives caused by nonspecific amplification. More importantly, its trans-cleavage activity is a key means of improving sensitivity and a crucial mechanism for signal output. For example, RPA isothermal amplification combined with the CRISPR / Cas12a system is used to detect African swine fever, and a matching PCR tube has been constructed to address the aerosol contamination issue associated with RPA. The synergy between efficient isothermal amplification and CRISPR / Cas12a has achieved a detection limit as low as 4 copies. Furthermore, using LAMP to amplify the target and CRISPR / Cas12a to recognize the amplified product significantly improves sensitivity, achieving a detection limit as low as 2 copies. Therefore, the above demonstrates that, with the assistance of the CRISPR / Cas12a system, detection methods can significantly improve both sensitivity and specificity. Therefore, CRISPR / Cas12a is a promising and valuable method.

[0007] Currently, there are no CaPV typing detection methods that combine array-based methods with CRISPR / Cas12a. Therefore, a CaPV typing detection method based on array-based methods and CRISPR / Cas12a has been developed, which is of great significance for rapid on-site inspections at customs ports, farms, and grassroots animal disease prevention and control agencies. Summary of the Invention

[0008] In order to fill the technical gap of the lack of accurate and sensitive CaPV typing and overcome the shortcomings of the existing technology, the first purpose of the present invention is to provide a CaPV typing detection method based on array mode and CRISPR / Cas12a, the second purpose is to use a constant temperature fluorescence instrument to realize the rapid identification of CaPV typing detection results, and the third purpose is to use a portable test strip to realize a point-of-care method for CaPV typing.

[0009] In order to achieve the above objectives, the technical solution adopted by the present invention is: a capripoxvirus typing detection kit based on array mode and CRISPR / Cas12a, comprising two pairs of primers and three crRNA sequences, the sequences of which are shown below:

[0010] B22R-Forward primer: 5'-CTTTTTCCATGATATGGTGGGC-3'

[0011] B22R-Reverse primer: 5'-TGAATGTGATCTCATATCCTTATTG-3'

[0012] RPO 30-Forward primer: 5'-TCTATGTCTTGATATGTGGTGGTAG-3'

[0013] RPO 30-Reverse primer: 5'-AGTGATTAGGTGGTGTATTATTTTCC-3'

[0014] crRNA1 sequence:

[0015] 5'-AAUUUCUACUAAGUGUAGAUUAAAAAAAGAAAAAAAAAAAG-3'

[0016] crRNA2 sequence:

[0017] 5'-AAUUUCUACUAAGUGUAGAUAUACAAAUAAAUAAAUUGCUU-3'

[0018] crRNA3 sequence:

[0019] 5'-AAUUUCUACUAAGUGUAGAUUACUGAAGUUGUUUAUUUUCA-3'.

[0020] The kit also includes ddH2O, 2×Ex Taq (Probe qPCR) MIX, LbCas12a, ssDNA-FQ reporter and 10×NEB buffer 2.1.

[0021] When performing capripoxvirus typing testing, there are two systems involved:

[0022] PCR system: 2×Ex Taq (Probe qPCR) MIX 12.5μL, 1μL 10μM B22R-Forward primer and B22R-Reverse primer, or RPO 30-Forward primer and RPO 30-Reverse primer, 8.5μL ddH2O, and 2μL of sample to be tested; amplification program: 95°C 5min; 35 cycles of: 95°C 10s, 55°C 15s, and 72°C 15s; finally, extension at 72°C for 10min; amplification of the B22R and RPO 30 genes, respectively.

[0023] CRISPR / Cas12a system: 4 μL of B22R amplification reaction solution was added to the CRISPR / Cas12a systems of crRNA1 and crRNA2, respectively; 4 μL of RPO 30 gene amplification solution was added to the CRISPR / Cas12a system of crRNA3; CRISPR / Cas12a contained 2 μL of 1 μM LbCas12a, 2 μL of 1 μM crRNA, 1 μL of 10 μM ssDNA-FQ reporter, 2 μL of 10×NEB buffer 2.1, and 9 μL of ddH2O; the crRNA was a crRNA1 sequence, a crRNA2 sequence, or a crRNA3 sequence, and CRISPR / Cas12a systems containing crRNA1, crRNA2, and crRNA3 were constructed, respectively.

[0024] ssDNA-FQ reporter was used as the beacon molecule, and the final system was incubated on a constant temperature fluorescence instrument at 37°C for 15 min to collect signals. The results were judged as follows: if crRNA1, crRNA2, and crRNA3 all had signals, it was LSDV; if only crRNA2 had a signal, it was SPPV; if crRNA2 and crRNA3 had signals but crRNA1 had no signal, it was GTPV.

[0025] The array-based CRISPR / Cas12a-based capripoxvirus typing test strip kit includes a lateral flow test strip, two pairs of primers, and three crRNA sequences. The sequences are as follows:

[0026] B22R-Forward primer: 5'-CTTTTTCCATGATATGGTGGGC-3'

[0027] B22R-Reverse primer: 5'-TGAATGTGATCTCATATCCTTATTG-3'

[0028] RPO 30-Forward primer: 5'-TCTATGTCTTGATATGTGGTGGTAG-3'

[0029] RPO 30-Reverse primer: 5'-AGTGATTAGGTGGTGTATTATTTTCC-3'

[0030] crRNA1 sequence:

[0031] 5'-AAUUUCUACUAAGUGUAGAUUAAAAAAAGAAAAAAAAAAAG-3'

[0032] crRNA2 sequence:

[0033] 5'-AAUUUCUACUAAGUGUAGAUAUACAAAUAAAUAAAUUGCUU-3'

[0034] crRNA3 sequence:

[0035] 5'-AAUUUCUACUAAGUGUAGAUUACUGAAGUUGUUUAUUUUCA-3'.

[0036] The kit also includes ddH2O, 2×Ex Taq (Probe qPCR) MIX, LbCas12a, ssDNA-LFA reporter (FAM / Bio) and 10×NEB buffer 2.1.

[0037] When performing capripoxvirus typing testing, there are two systems involved:

[0038] PCR system: 2×Ex Taq (Probe qPCR) MIX 12.5μL, 1μL 10μM B22R-Forward primer and B22R-Reverse primer, or RPO 30-Forward primer and RPO 30-Reverse primer, 8.5μL ddH2O, and 2μL of sample to be tested; amplification program: 95°C 5min; 35 cycles of: 95°C 10s, 55°C 15s, and 72°C 15s; finally, extension at 72°C for 10min; amplification of the B22R and RPO 30 genes, respectively.

[0039] CRISPR / Cas12a system: 4 μL of B22R amplification reaction solution was added to the CRISPR / Cas12a systems of crRNA1 and crRNA2, respectively; 4 μL of RPO 30 gene amplification solution was added to the CRISPR / Cas12a system of crRNA3; CRISPR / Cas12a contained 2 μL of 1 μM LbCas12a, 2 μL of 1 μM crRNA, 1 μL of 10 μM ssDNA-LFA reporter, 2 μL of 10×NEB buffer 2.1, and 9 μL of ddH2O; the crRNA was a crRNA1 sequence, a crRNA2 sequence, or a crRNA3 sequence, and CRISPR / Cas12a systems containing crRNA1, crRNA2, and crRNA3 were constructed, respectively.

[0040] After the CRISPR / Cas12a system reaction is completed, the test strip is inserted into ddH2O and placed at room temperature for 2 minutes. The results are judged as follows: the appearance of both T lines and C lines or only T lines is positive, and only C lines are negative. Among them, if the crRNA1, crRNA2 and crRNA3 systems all have T lines, it is LSDV; if only crRNA2 has a T line, it is SPPV; if crRNA2 and crRNA3 have T lines but crRNA1 has no T line, it is GTPV.

[0041] The application of the above-mentioned array mode and CRISPR / Cas12a-based capripoxvirus typing detection kit and test strip kit in the preparation of capripoxvirus typing detection supplies.

[0042] When using the above kit for detection, the following steps are included:

[0043] (1) PCR: Add the sample to be tested into the above PCR system to amplify the B22R and RPO 30 genes respectively;

[0044] (2) Array CRISPR / Cas12a reaction: 4 μL of the reaction solution from step (1) was added to each of the three CRISPR / Cas12a reaction systems mentioned above and reacted at 37°C for 15 min;

[0045] (3) Result determination

[0046] i. CRISPR / Cas12a-mediated array fluorescence typing detection: If crRNA1, crRNA2, and crRNA3 systems all have signals, it is LSDV; if only crRNA2 has a signal, it is SPPV; if crRNA2 and crRNA3 have signals but crRNA1 has no signal, it is GTPV.

[0047] ii. Lateral flow test based on CRISPR / Cas12a: Insert the test strip into the reaction solution (2) above (ssDNA-LFA reporter modified with FAM and Biotin as beacon molecules) and place at room temperature for 2 minutes. If both T lines and C lines appear or only T lines appear, it is positive, while only C lines appear, which is negative. Among them, if crRNA1, crRNA2, and crRNA3 systems all have T lines, it is LSDV; if only crRNA2 has T lines, it is SPPV; if crRNA2 and crRNA3 have T lines but crRNA1 has no T lines, it is GTPV.

[0048] The principle of the present invention is that: in the B22R gene, LSDV adds a 34-36bp fragment, while SPPV and GTPV do not add it; and in the RPO 30 gene, SPPV deletes a 21bp fragment, while LSDV and GTPV do not delete it. We constructed an array mode sensor based on PCR combined with CRISPR / Cas12a system for typing research. Figure 1 (A) Two genes are amplified by PCR, and the resulting amplicons are used for CRISPR / Cas12a system recognition. crRNA1 targets the 34-36bp added to the B22R gene of LSDV. The binary complex formed by crRNA1 and Cas12a recognizes the target through the PAM site and crRNA1 complements this sequence, thereby activating the cis and trans cleavage activities of Cas12a. The cis cleavage activity will cleave the amplicon, while the trans cleavage activity will cleave the large number of single-stranded reporter probes (ssDNA-FQ reporter) around it. This moves the fluorescent group on the reporter probe away from the quenching group, thereby emitting fluorescence. crRNA2 is designed to be complementary to the common sequence of LSDV, SPPV and GTPV, and is used to detect all genotypes. CrRNA3 targets the 21 bp deleted in the RPO 30 gene of SPPV. In other words, SPPV does not produce fluorescence due to the deletion, while LSDV and GTPV have strong fluorescence. Therefore, LSDV has three crRNAs that produce fluorescence; SPPV has only crRNA2 that produces fluorescence; and GTPV has crRNA2 and crRNA3 that produce fluorescence. This array format, used for typing detection, greatly improves detection accuracy.

[0049] In order to achieve visual detection and facilitate detection in areas where there is a lack of fluorescence instruments, we use lateral flow test strips for typing detection. Figure 1(B) Anti-FAM antibodies are attached to Au nanoparticles, streptavidin is coated on line C, and a secondary antibody is contained on line T. As the liquid flows, the FAM on the reporter probe connects to the Au nanoparticles through the binding of FAM and anti-FAM antibodies. The intact reporter probe is intercepted on line C through the binding of streptavidin and biotin, appearing as a red line on line C. After the reporter probe is cleaved, the biotin-attached DNA fragments, including the truncated reporter probe and the intact reporter probe, are intercepted on line C, while the broken DNA attached to the anti-FAM-Au nanoparticles is intercepted on line T through the secondary antibody, resulting in a red line on line T. Using the lateral flow test strip, typing results can be directly observed with the naked eye. The array test strips accurately type LSDV, SPPV, and GTPV.

[0050] The advantages of the present invention are: (1) the array mode is more accurate for typing detection, which can avoid misjudgment caused by errors in a certain step; (2) accurate typing detection makes the selection of control and eradication measures more targeted; (3) high sensitivity: the efficient amplification of PCR combined with the signal amplification ability of the trans-cleavage activity of CRISPR / Cas12a makes the method highly sensitive, with a time detection limit as low as 50, 40, 60 copies for LSDV, SPPV and GTPV. High sensitivity can solve the false negative phenomenon caused by low abundance. (4) high specificity: the high specificity recognition of crRNA of the CRISPR system combined with the multiple discrimination of the array, the positive detection rate reaches 100%; (5) simple identification: the test strip test results can be identified by the naked eye; (6) wide application: it can be widely used for CaPV typing detection; (7) convenient: the use of a portable fluorimeter and a test strip can achieve instant detection (POCT) of CaPV. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 It is based on the principle of CaPV typing detection in combination with the CRISPR / Cas12a system in array mode;

[0052] Figure 2 This is a feasibility study of using array sensors for typing; (A) Detection of three viruses by the crRNA1 system; (B) Detection of three viruses by the crRNA2 system; (C) Detection of three viruses by the crRNA3 system; (D) Detection of three viruses by the array test strip system based on three CRISPR / Cas12a systems; (E) Imaging of the results of the fluorescent array sensor in a fluorescent imager;

[0053] Figure 3Validation of the specificity of array sensors for typing; (A) Detection of 7 viruses by the crRNA1 system; (B) Detection of 7 viruses by the crRNA2 system; (C) Detection of 7 viruses by the crRNA3 system; (D) Detection of 7 viruses by the array test strip system based on three CRISPR / Cas12a systems;

[0054] Figure 4 Figure 1 is the sensitivity of the array sensor for LSDV detection; (A) is the fluorescence spectrum of the crRNA1 system detecting LSDV virus at different concentrations; (B) is the fluorescence spectrum of the crRNA2 system detecting LSDV virus at different concentrations; (C) is the fluorescence spectrum of the crRNA3 system detecting LSDV virus at different concentrations; (D) is the result of the array test strip system detecting LSDV virus at different concentrations based on three CRISPR / Cas12a systems;

[0055] Figure 5 The sensitivity of the array sensor for SPPV detection; (A) is the fluorescence spectrum of the crRNA1 system for detecting SPPV viruses at different concentrations; (B) is the fluorescence spectrum of the crRNA2 system for detecting SPPV viruses at different concentrations; (C) is the fluorescence spectrum of the crRNA3 system for detecting SPPV viruses at different concentrations; (D) is the result of the array test strip system detecting SPPV viruses at different concentrations based on three CRISPR / Cas12a systems;

[0056] Figure 6 The sensitivity of the array sensor for GTPV detection; (A) is the fluorescence spectrum of the crRNA1 system detecting GTPV viruses at different concentrations; (B) is the fluorescence spectrum of the crRNA2 system detecting GTPV viruses at different concentrations; (C) is the fluorescence spectrum of the crRNA3 system detecting GTPV viruses at different concentrations; (D) is the result of the array test strip system detecting GTPV viruses at different concentrations based on three CRISPR / Cas12a systems;

[0057] Figure 7 It is a heat map analysis of the detection results of 50 real samples by the fluorescence array sensor;

[0058] Figure 8 It is the test result of 50 actual samples by lateral flow test strip array sensor;

[0059] Figure 9Figure 1 is the fluorescence array sensor's analysis of actual samples 1-16; (A) (B) and (C) are the fluorescence spectra of samples 1-16 under the crRNA1, crRNA2, and crRNA3 systems; (D) is the test strip array's analysis of samples 1-16;

[0060] Figure 10 Figure 1 is the fluorescence array sensor's analysis of actual samples 17-34; (A) (B) and (C) are the fluorescence spectra of samples 17-34 under the crRNA1, crRNA2, and crRNA3 systems; (D) is the test strip array's analysis of samples 17-34.

[0061] Figure 11 Figure 3 is the detection and analysis of actual samples 35-50 by the fluorescence array sensor; (A) (B) and (C) are the fluorescence spectra of samples 35-50 under the crRNA1, crRNA2 and crRNA3 systems; (D) is the detection of samples 35-50 by the test strip array. DETAILED DESCRIPTION

[0062] Example 1: According to the above principle analysis, the sequences of primers and crRNA shown in the following table were designed.

[0063]

[0064]

[0065] The capripoxvirus (CaPV) typing detection method based on the array mode combined with the CRISPR / Cas12a system is as follows, including two systems:

[0066] PCR system:

[0067] 2×Ex Taq (Probe qPCR) mix: 12.5 μL, 1 μL of 10 μM B22R-Forward and B22R-Reverse primers, or RPO 30-Forward and RPO 30-Reverse primers, 8.5 μL of ddH2O, and 2 μL of target. Amplification program: 95°C for 5 min; 35 cycles of 95°C for 10 s, 55°C for 15 s, and 72°C for 15 s; finally, extension at 72°C for 10 min. The two primer sets amplify the B22R and RPO 30 genes, respectively.

[0068] CRISPR / Cas12a system:

[0069] 4 μL of the B22R amplification reaction solution was added to the CRISPR / Cas12a systems for crRNA1 and crRNA2, respectively. 4 μL of the RPO 30 gene amplification solution was added to the CRISPR / Cas12a system for crRNA3. The CRISPR / Cas12a system contains 2 μL of 1 μM LbCas12a, 2 μL of 1 μM crRNA, 1 μL of a 10 μM ssDNA-FQ reporter (for fluorescence detection) or 1 μL of a 10 μM ssDNA-LFA reporter (for test strip detection), 2 μL of 10× NEB buffer 2.1, and 9 μL of ddH2O.

[0070] Array Mode:

[0071] There are three different crRNAs designed for three specific regions, namely crRNA1, crRNA2 and crRNA3. Therefore, the three CRISPR / Cas12a systems mentioned above each add three crRNAs for reaction to form an array pattern.

[0072] For CRISPR / Cas12a-mediated fluorescence detection, ssDNA-FQ reporter was used as the beacon molecule, and the final system was incubated on a constant temperature fluorescence instrument at 37°C for 15 min to collect the signal.

[0073] A CRISPR / Cas12a-based lateral flow test strip uses an ssDNA-LFA reporter as a beacon molecule. After the CRISPR / Cas12a system completes the reaction, 80 μL of ddH2O is added. The test strip is inserted and left at room temperature for 2 minutes to perform point-of-care CaPV typing.

[0074] Result judgment:

[0075] i. CRISPR / Cas12a-mediated array fluorescence typing detection: If crRNA1, crRNA2, and crRNA3 systems all have signals, it is LSDV; if only crRNA2 has a signal, it is SPPV; if crRNA2 and crRNA3 have signals but crRNA1 has no signal, it is GTPV.

[0076] ii. Lateral flow test based on CRISPR / Cas12a: Insert the test strip into the reaction solution (2) above (ssDNA-LFA reporter as beacon molecule) and place at room temperature for 2 minutes. If both T lines and C lines appear or only T lines appear, it is positive, while only C lines appear, which is negative. Among them, if the crRNA1, crRNA2, and crRNA3 systems all have T lines, it is LSDV; if only crRNA2 has T lines, it is SPPV; if crRNA2 and crRNA3 have T lines but crRNA1 does not have T lines, it is GTPV.

[0077] Example 2, feasibility verification:

[0078] In order to verify the feasibility of the method for CaPV typing, three crRNAs were tested for LSDV, SPPV and GTPV. Figure 2 In the detection of crRNA1, only LSDV showed significant fluorescence, while the others showed no fluorescence. When these were detected using crRNA2, they showed strong fluorescence, far exceeding that of the control group. Under the action of crRNA3, LSDV and GTPV showed strong fluorescence, while SPPV showed no fluorescence. These phenomena are consistent with theory: all three crRNAs of LSDV showed significant fluorescence; SPPV showed fluorescence only under the action of crRNA3; and GTPV showed significant fluorescence in crRNA2 and crRNA3. This also demonstrates the excellent specificity of CRISPR / Cas12a for typing detection. In addition, using a fluorescence imager to detect each unit in the array, it was found that LSDV showed fluorescence in all three crRNAs, while only crRNA2 of SPPV showed fluorescence, and GTPV showed fluorescence in crRNA2 and crRNA3.

[0079] In the test strip test, it is obvious that LSDV has bands on the T line under all three crRNAs; SPPV has a band only in crRNA2, and no other bands on the T line; while GTPV has bands on the T line in the detection of crRNA2 and crRNA3. Figure 2 (D) This result is consistent with the fluorescence detection result, indicating that the CRISPR / Cas12a system combined with test strip detection is very feasible for typing.

[0080] Example 3, specificity verification

[0081] Four viruses that commonly infect cattle and sheep were selected as interference groups to explore the specificity of PCR and CRISPR / Cas12a. Figure 3In the crRNA1 detection system, only LSDV has obvious fluorescence, while the others have no fluorescence. In crRNA2, LSDV, SPPV and GTPV have fluorescence, while the other four have no fluorescence, indicating that the B22R primer is very specific to the CaPV virus; in the crRNA3 detection system, only LSDV and GTPV produce fluorescence, while the others have no fluorescence, indicating that the RPO 30 primer and crRNA have strong specificity for the CaPV virus and are suitable for typing detection. The participation of CRISPR / Cas12a greatly enhances the specificity of the method through the recognition of amplicons by crRNA, which also solves the false positive problem caused by nonspecific amplification. By using lateral flow test strips for detection, Figure 3 (D) shows consistent results. The above results indicate that the array sensor based on dual PCR combined with CRISPR / Cas12a has strong specificity in CaPV typing detection.

[0082] Example 4, method sensitivity verification

[0083] Under the action of PCR, the target is amplified. In addition, the trans-cleavage activity of CRISPR / Cas12a makes a great contribution to the sensitivity. Figure 4 , different concentrations of LSDV were reacted in three crRNA systems. It was found that the fluorescence intensity decreased with decreasing concentration. When the concentration was as low as 25 copies, the fluorescence of crRNA1 and crRNA2 was the lowest, while in the crRNA3 system, when the concentration was as low as 50 copies, the fluorescence dropped to the lowest. This may be related to the amplification ability of the primers and the pairing ability of crRNA. Therefore, in the detection of LSDV, the actual detection limit of the array mode method was as low as 50 copies. Similarly, SPPV reacted in three crRNA systems. Figure 5 In crRNA1 and crRNA3, there is no fluorescence intensity regardless of the concentration. In crRNA2, the fluorescence intensity increases with the increase of concentration, indicating that the fluorescence intensity is dependent on the target concentration. As the number of targets increases, the number of amplicons increases, which can activate more Cas12a to react, and the fluorescence intensity also increases. Therefore, the detection limit of SPPV depends on the detection limit of crRNA2, which is 40 copies. Finally, the array's detection ability for GTPV is reflected in Figure 6In crRNA1, no fluorescence is generated. In crRNA2 and crRNA3, the fluorescence intensity increases with increasing concentration. Under the action of crRNA2, the detection limit is as low as 60 copies, and under the action of crRNA3, the detection limit is as low as 30 copies. In summary, the detection limit of the array sensor for GTPV is as low as 60 copies. Compared with the melting curve method for typing detection, the sensitivity of the present invention is higher. High sensitivity increases the accuracy of detection and does not require expensive instruments. Compared with the method of PCR combined with agarose gel electrophoresis, the sensitivity of the sensor of the present invention is superior. More importantly, the detection time of this method is short, and it is completed within 1 hour and 15 minutes, which provides a fast, sensitive and accurate method for typing detection.

[0084] In the test strip detection, their sensitivity is slightly lower than the fluorescence signal. This is determined by the nature of the colorimetric visualization detection based on Au nanoparticles. Figure 4 In (D), the actual detection limit of the crRNA1 detection system is as low as 500 copies, while the actual detection limit of crRNA2 and crRNA3 is as low as 50 copies. In summary, the actual detection limit of the test strip for LSDV is as low as 500 copies. Figure 5 In (D), the array test strips were used to detect SPPV. It was found that no matter the concentration, the test strips of crRNA1 and crRNA3 had no T line, and only crRNA2 had a T line. The detection limit was as low as 800 copies. Finally, the test strip array was used to detect SPPV. Figure 6 (D) It was found that there were no T lines in crRNA1, but there were obvious T lines in crRNA2 and crRNA3, and the detection limit was as low as 300 copies in crRNA2 and 600 copies in crRNA3. Taken together, the actual detection limit of the array test strip for GTPV was as low as 600 copies.

[0085] Example 5: Actual sample detection

[0086] In order to verify the detection ability of the array typing sensor in actual samples, 50 samples including throat swabs, nasal swabs, blood and tissues from cattle and sheep were used for detection, among which 1-16 samples were from cattle, 17-34 samples were from sheep, and 35-50 samples were from goats. Figure 7 , 1, 2, 3, 4, 6, 7, 8, 9 all have obvious fluorescence in the three crRNA detection systems, they are LSDV (fluorescence spectrum see Figure 9 ); Among 17, 18, 19, 20, 21, 23, 28 and 32, only crRNA2 has fluorescence, and they are SPPV (fluorescence spectrum see Figure 10Finally, samples 35, 36, 37, 40, 43, 44, 45, 46, 47, 48, and 49 all had significant fluorescence in crRNA2 and crRNA3, but no fluorescence in crRNA1, so they were identified as GTPV (fluorescence spectra are shown in Figure 11 ). The rest were negative. Moreover, they were consistent with the source, which also indicates that there is no cross-virus infection in these samples. In addition, in order to determine whether they are CaPV viruses, industry-standard primers and probes were used to verify the samples, such as Figure 9 (D), 10(D) and 11(D) found that their results were consistent with the results of the array typing sensor. In addition, 50 samples were tested using lateral flow test strips and found that they had the same results. Figure 8 Therefore, the accuracy of the lateral flow test strip is very high. In summary, the array typing sensor of the present invention has good detection capabilities in practical applications.

Claims

1. A capripoxvirus typing detection kit based on array format and CRISPR / Cas12a, characterized by: It includes two pairs of primers and three crRNA sequences, the sequences of which are as follows: B22R-Forward primer: 5'-CTTTTTCCATGATATGGTGGGC-3' B22R-Reverse primer: 5'-TGAATGTGATCTCATATCCTTATTG-3' RPO 30-Forward primer: 5'-TCTATGTCTTGATATGTGGTGGTAG-3' RPO 30-Reverse primer: 5'-AGTGATTAGGTGGTGTATTATTTTCC-3' crRNA1 sequence: 5'-AAUUUCUACUAAGUGUAGAUUAAAAAAAGAAAAAAAAAAAG-3' crRNA2 sequence: 5'-AAUUUCUACUAAGUGUAGAUAUACAAAUAAAUAAAUUGCUU-3' crRNA3 sequence: 5'-AAUUUCUACUAAGUGUAGAUUACUGAAGUUGUUUAUUUUCA-3' When performing capripoxvirus typing testing, there are two systems involved: PCR system: 2×Ex Taq (Probe qPCR) MIX 12.5 μL, 1 μL 10 μM B22R-Forward primer and B22R-Reverse primer, or RPO 30-Forward primer and RPO 30-Reverse primer, 8.5 μL ddH2O, and 2 μL test sample; amplification program: 95°C for 5 min; 35 cycles of 95°C for 10 s, 55°C for 15 s, and 72°C for 15 s; finally, extension at 72°C for 10 min; amplification of the B22R and RPO 30 genes, respectively; CRISPR / Cas12a system: 4 μL of B22R amplification reaction solution was added to the CRISPR / Cas12a systems of crRNA1 and crRNA2, respectively; 4 μL of RPO 30 gene amplification solution was added to the CRISPR / Cas12a system of crRNA3; CRISPR / Cas12a contained 2 μL of 1 μM LbCas12a, 2 μL of 1 μM crRNA, 1 μL of 10 μM ssDNA-FQ reporter, 2 μL of 10×NEB buffer 2.1, and 9 μL of ddH2O; the crRNA was a crRNA1 sequence, a crRNA2 sequence, or a crRNA3 sequence, and CRISPR / Cas12a systems containing crRNA1, crRNA2, and crRNA3 were constructed, respectively.

2. The capripoxvirus typing detection kit based on array mode and CRISPR / Cas12a according to claim 1, characterized in that: It also includes ddH2O, 2×Ex Taq (Probe qPCR) MIX, LbCas12a, ssDNA-FQ reporter and 10×NEB buffer 2.

1.

3. The capripoxvirus typing detection kit based on array mode and CRISPR / Cas12a according to claim 1, characterized in that: ssDNA-FQ reporter was used as the beacon molecule, and the final system was incubated on a constant temperature fluorescence instrument at 37°C for 15 minutes to collect signals. The results were determined as follows: if crRNA1, crRNA2, and crRNA3 all had signals, it was LSDV; if only crRNA2 had a signal, it was SPPV; if crRNA2 and crRNA3 had signals but crRNA1 had no signal, it was GTPV.

4. Use of the capripoxvirus typing detection kit based on array mode and CRISPR / Cas12a according to any one of claims 1 to 3 in the preparation of capripoxvirus typing detection supplies.

5. A capripoxvirus typing test strip kit based on array format and CRISPR / Cas12a, including a lateral flow test strip, characterized by: It also includes two pairs of primers and three crRNA sequences, the sequences of which are as follows: B22R-Forward primer: 5'-CTTTTTCCATGATATGGTGGGC-3' B22R-Reverse primer: 5'-TGAATGTGATCTCATATCCTTATTG-3' RPO 30-Forward primer: 5'-TCTATGTCTTGATATGTGGTGGTAG-3' RPO 30-Reverse primer: 5'-AGTGATTAGGTGGTGTATTATTTTCC-3' crRNA1 sequence: 5'-AAUUUCUACUAAGUGUAGAUUAAAAAAAGAAAAAAAAAAAG-3' crRNA2 sequence: 5'-AAUUUCUACUAAGUGUAGAUAUACAAAUAAAUAAAUUGCUU-3' crRNA3 sequence: 5'-AAUUUCUACUAAGUGUAGAUUACUGAAGUUGUUUAUUUUCA-3' When performing capripoxvirus typing detection, two systems are included: PCR system: 2×Ex Taq (Probe qPCR) MIX 12.5 μL, 1 μL 10 μM B22R-Forward primer and B22R-Reverse primer, or RPO 30-Forward primer and RPO 30-Reverse primer, 8.5 μL ddH2O, and 2 μL test sample; amplification program: 95°C for 5 min; 35 cycles of 95°C for 10 s, 55°C for 15 s, and 72°C for 15 s; finally, extension at 72°C for 10 min; amplification of the B22R and RPO 30 genes, respectively; CRISPR / Cas12a system: 4 μL of B22R amplification reaction solution was added to the CRISPR / Cas12a systems of crRNA1 and crRNA2, respectively; 4 μL of RPO 30 gene amplification solution was added to the CRISPR / Cas12a system of crRNA3; CRISPR / Cas12a contained 2 μL of 1 μM LbCas12a, 2 μL of 1 μM crRNA, 1 μL 10 μM ssDNA-LFA reporter, 2 μL 10×NEB buffer 2.1 and 9 μL ddH2O; the crRNA was a crRNA1 sequence, a crRNA2 sequence or a crRNA3 sequence, and CRISPR / Cas12a systems containing crRNA1, crRNA2 and crRNA3 were constructed respectively.

6. The capripoxvirus typing test strip kit based on array mode and CRISPR / Cas12a according to claim 5, characterized in that: Also included are ddH2O, 2×Ex Taq (Probe qPCR) MIX, LbCas12a, FAM / Bioreporter, and 10×NEB buffer 2.

1.

7. The capripoxvirus typing test strip kit based on array mode and CRISPR / Cas12a according to claim 5, characterized in that: After the CRISPR / Cas12a system reaction is completed, the test strip is inserted into ddH2O and placed at room temperature for 2 minutes. The results are judged as follows: the appearance of both T lines and C lines or only T lines is positive, and only C lines are negative. Among them, if the crRNA1, crRNA2 and crRNA3 systems all have T lines, it is LSDV; if only crRNA2 has a T line, it is SPPV; if crRNA2 and crRNA3 have T lines but crRNA1 has no T line, it is GTPV.

8. Use of the capripoxvirus typing test strip kit based on array mode and CRISPR / Cas12a according to any one of claims 5 to 7 in the preparation of capripoxvirus typing detection supplies.

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