Multiplex nucleic acid detection technology based on CRISPR-Gs12 system mediation and application

Abstract

The invention provides a multiple nucleic acid detection technology based on CRISPR / Gs12 system mediation and application, different Gs12 nuclease are respectively combined with corresponding specific crRNA and single-stranded DNA reporter genes, and then a recombinase polymerase amplification (RPA) method is combined, so that quadruple, triple or double specific accurate detection of target nucleic acid molecules can be realized. Therefore, a nucleic acid detection technology and a kit capable of simultaneously distinguishing the Seneca virus (SVA), the hog cholera virus (CSFV) and the African hog cholera virus (ASFV) are developed. The method provided by the invention can realize rapid multiple detection of target nucleic acid, and has good popularization and application values in the field of nucleic acid detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and specifically relates to a CRISPR-Gs12 system-mediated multiple nucleic acid detection technology and its application. Background Art

[0002] Clusters of regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR / Cas) are considered to be the next generation of molecular diagnostic tools. It is an adaptive immune system of bacteria and archaea, and the OMEGARNA programmable system reported later may be the ancestor of CRISPR / Cas. Recent studies have also described Fanzor nucleases, which may belong to the eukaryotic editing system and are the evolutionary products of bacterial infection of eukaryotic organisms. The above situation shows that in the process of biological evolution, the high evolution of the host, composition, structure and function of Cas nucleases has led to differences in nucleic acid targets, sequence preferences and binding activities. Since the V-type Cas12a family and the VI-type Cas13a family have unique cutting activity and excellent compatibility, guided by a single CRISPR RNA (crRNA) complementary to the target sequence, without the need for a separate trans-activating crRNA (tracRNA), the CRISPR / Cas system plays an important role in nucleic acid diagnosis.

[0003] At present, the infection and spread of pathogens remain one of the major threats to public health. In pathogen nucleic acid diagnosis, the detection of a single viral nucleic acid in an infected individual is not sufficient for accurate diagnosis in the early stages of viral infection, especially in agricultural environments such as pig farms, where multiple viruses are easily infected at the same time due to immune deficiency. In addition, the detection of multiple different markers in the same infected individual is cumbersome and time-consuming. Therefore, it is necessary to develop multiple nucleic acid detection technologies to detect multiple nucleic acids simultaneously in an accurate, economical and efficient manner, and parallel multiple detection is the trend of the development of diagnostic methods and equipment. Using Cas effectors coupled with biosensing devices and molecular elements, many highly sensitive and parallel multiple detection methods have been developed, which require operators with multidisciplinary backgrounds and equipment that is mostly limited to laboratory environments. The strategy of using CRISPR / Cas12 and Cas13 combined detection shows great promise in the development of highly sensitive and rapid multiple pathogen nucleic acid diagnostic assays, and the use of fluorescent groups and quenched molecular beacons to read the results is also more suitable for POCT. However, Cas13 can only recognize RNA targets, the diagnostic process involves reverse transcription, and the instability of RNA will bring difficulties to the reaction, limiting the scope of application. Therefore, it is urgent to develop new technologies for multiple nucleic acid detection mediated by the Cas12a system alone.

[0004] The present invention proposes a method for realizing multiple nucleic acid detection based on CRISPR / Cas12a family homologous nucleases. First, we screened 4 nucleases Gs12-1, Gs12-4, Gs12-9 and Gs12-18 from the newly discovered Cas12a homologous nucleases. Comparison found that they have specific base preferences for ssDNA-FQ reporter genes. In addition, by truncating, adding or modifying the secondary structure of crRNA, and by changing the scaffold sequence composition in crRNA, the best crRNA variant that can improve the trans-cutting specificity of Gs12 nuclease was identified. Three non-interfering fluorescent modification groups were further screened and determined, and high sensitivity and specific fluorescence detection of three viral nucleic acids of Seneca virus (SVA), classical swine fever virus (CSFV) and African swine fever virus (ASFV) were achieved by combining multiple RPA amplification, as well as naked eye visualization detection of two viral nucleic acids of SVA and ASFV.

[0005] In summary, the present invention expands the application scope of Cas12a family nucleases, and for the first time uses Cas12 family nuclease members to realize multiple nucleic acid detection, providing a new means for the development of multiple nucleic acid detection technology mediated by the CRISPR system. Summary of the invention

[0006] The purpose of the present invention is to provide a CRISPR / Gs12 system-mediated multiple nucleic acid detection technology and application.

[0007] The first aspect of the present invention provides a multiple nucleic acid detection kit mediated by the CRISPR / Gs12 system.

[0008] A multiple nucleic acid detection kit based on CRISPR / Gs12 system, comprising:

[0009] (1) Gs12 nuclease, wherein the Gs12 nuclease comprises at least two of Gs12-1, Gs12-4, Gs12-9 and Gs12-18 nucleases, wherein the amino acid sequence of Gs12-1 is shown in SEQ ID NO.1, the amino acid sequence of Gs12-4 is shown in SEQ ID NO.4, the amino acid sequence of Gs12-9 is shown in SEQ ID NO.9, and the amino acid sequence of Gs12-18 is shown in SEQ ID NO.18;

[0010] (2) A reporter gene specifically corresponding to the Gs12 nuclease, wherein the reporter gene sequence corresponding to the Gs12-1 nuclease is 5′-AAAAA-3′, the reporter gene sequence corresponding to the Gs12-4 nuclease is 5′-TTTTT-3′, the reporter gene sequence corresponding to the Gs12-9 nuclease is 5′-TATAT-3′, and the reporter gene sequence corresponding to the Gs12-18 nuclease is 5′-TTATT-3′, and the 5′ end of the reporter gene is modified with a fluorescent group whose excitation wavelengths do not interfere with each other, and the 3′ end is modified with a quenching group;

[0011] (3) a cRNA corresponding to the Gs12 nuclease, the cRNA containing a scaffold and a targeting sequence; preferably, the scaffold sequence in the crRNA of the Gs12-1 nuclease is shown in SEQ ID NO.22, the scaffold sequence in the crRNA of the Gs12-4 nuclease is shown in SEQ ID NO.23, the scaffold sequence in the crRNA of the Gs12-9 nuclease is shown in SEQ ID NO.24, and the scaffold sequence in the crRNA of the Gs12-18 nuclease is shown in SEQ ID NO.25.

[0012] The second aspect of the present invention provides a multiple nucleic acid detection kit for Seneca virus, classical swine fever virus and African swine fever virus mediated by the CRISPR / Gs12 system.

[0013] The kit for multiplex detection of Seneca virus, classical swine fever virus and African swine fever virus mediated by CRISPR / Gs12 system includes:

[0014] (1) Gs12 nuclease, wherein the Gs12 nuclease is selected from any three of Gs12-1, Gs12-4, Gs12-9 and Gs12-18 nucleases, wherein the amino acid sequence of Gs12-1 is shown in SEQ ID NO.1, the amino acid sequence of Gs12-4 is shown in SEQ ID NO.4, the amino acid sequence of Gs12-9 is shown in SEQ ID NO.9, and the amino acid sequence of Gs12-18 is shown in SEQ ID NO.18; preferably, the Gs12 nuclease is the three nucleases of Gs12-1, Gs12-4 and Gs12-18;

[0015] (2) A reporter gene specifically corresponding to the Gs12 nuclease, wherein the reporter gene sequence corresponding to the Gs12-1 protein is 5′-AAAAA-3′, the reporter gene sequence corresponding to the Gs12-4 protein is 5′-TTTTT-3′, the reporter gene sequence corresponding to the Gs12-9 protein is 5′-TATAT-3′, and the reporter gene sequence corresponding to the Gs12-18 protein is 5′-TTATT-3′, and the 5′ end of the reporter gene is modified with a fluorescent group whose excitation wavelengths do not interfere with each other, and the 3′ end is modified with a quenching group;

[0016] (3) cRNA specifically targeting the Seneca virus VP1 gene, the classical swine fever virus E2 gene and the African swine fever virus P72 gene, wherein the cRNA sequence targeting the Seneca virus VP1 gene is shown in SEQ ID NO.26, the cRNA sequence targeting the classical swine fever virus E2 gene is shown in SEQ ID NO.27, and the cRNA sequence targeting the African swine fever virus P72 gene is shown in SEQ ID NO.28.

[0017] Furthermore, the kit also includes primers for amplifying the Seneca virus VP1 gene, the classical swine fever virus E2 gene and the African swine fever virus P72 gene. Preferably, the RPA primer sequence for amplifying the Seneca virus VP1 gene is shown in SEQ ID NOs. 29 and 30, the RPA primer sequence for amplifying the classical swine fever virus E2 gene is shown in SEQ ID NOs. 31 and 32, and the RPA primer sequence for amplifying the African swine fever virus P72 gene is shown in SEQ ID NOs. 33 and 34.

[0018] In a third aspect, the present invention provides a kit for dual visualization detection of Seneca virus and African swine fever virus mediated by the CRISPR / Gs12 system.

[0019] The kit for dual visualization detection of Seneca virus and African swine fever virus based on CRISPR / Gs12 system includes:

[0020] (1) Gs12-1 and Gs12-18 nucleases, wherein the amino acid sequence of Gs12-1 is shown in SEQ ID NO.1, and the amino acid sequence of Gs12-18 is shown in SEQ ID NO.18;

[0021] (2) The reporter gene sequence corresponding to the Gs12-1 nuclease is 5′-AAAAA-3′, and the reporter gene sequence corresponding to the Gs12-18 nuclease is 5′-TTATT-3′, and the 5′ end of the reporter gene is modified with a fluorescent group FAM or ROX, and the 3′ end is modified with a quenching group;

[0022] (3) The cRNA sequence specifically targeting the Seneca virus VP1 gene is shown in SEQ ID NO.26, and the cRNA sequence specifically targeting the African swine fever virus P72 gene is shown in SEQ ID NO.28.

[0023] Furthermore, it also includes primers for amplifying the Seneca virus VP1 gene and the African swine fever virus P72 gene. Preferably, the RPA primer sequence for amplifying the Seneca virus VP1 gene is as shown in SEQ ID NOs. 29 and 30, and the RPA primer sequence for amplifying the African swine fever virus P72 gene is as shown in SEQ ID NOs. 33 and 34.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0025] 1. A multiplex nucleic acid detection technology mediated by the CRISPR / Gs12 system, which has the advantage of combining different Gs12 nucleases with their corresponding specific crRNA and single-stranded DNA reporter genes, and combining with multiple RPA amplification technology to achieve quadruple, triple or double specific detection of target nucleic acid molecules.

[0026] 2. A multiplex detection kit for Seneca virus, classical swine fever virus and African swine fever virus, which can simultaneously achieve accurate detection of three or two viral nucleic acid targets in one sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 .Shows the evaluation of the base preference of Gs12 nuclease for ssDNA-FQ reporter genes. A. Flowchart for screening the Cas12a family homologous nuclease Gs12; B. Phylogenetic tree of CRISPR / Gs12 family proteins; C. Detection of the base preference of different Gs12 nucleases for ssDNA-FQ reporter genes.

[0028] Figure 2.Shows the establishment of a multiplex nucleic acid detection technology mediated by the CRISPR-Gs12 system. A. Identification of the best combination of four Gs12 nucleases Gs12-1, Gs12-4, Gs12-9 and Gs12-18 and ssDNA-FQ reporter genes specifically modified with fluorescent groups; B. Detection of the trans-cleavage specificity of four Gs12 nucleases against specific ssDNA-FQ reporter genes; C. Improve the trans-cleavage specificity of Gs12 nucleases by screening different crRNA variants of scaffolds and determine the best combination; D. Screening of scaffold variants for the best crRNA paired with different Gs12 nucleases; E. Detection of the multiplex detection capability of the multiplex nucleic acid detection technology mediated by the CRISPR-Gs12 system for three targets, Target 1 represents the ASFV-P72 gene, Target 2 represents the SVA-VP1 gene, and Target 3 represents the CSFV-E2 gene.

[0029] Figure 3 .Shows the technology of triple nucleic acid detection of Seneca virus (SVA), classical swine fever virus (CSFV) and African swine fever virus (ASFV). A. Schematic diagram of triple nucleic acid detection mediated by the optimal combination of Gs12-1, Gs12-4, Gs12-18 proteins and ssDNA-FQ reporter gene; B. Evaluation of the specificity of CRISPR-Gs12s technology for simultaneous detection of three viruses; C. Flow chart of triple nucleic acid detection technology mediated by RPA-CRISPR / Gs12s system; D. Evaluation of the sensitivity of triple RPA-CRISPR / Gs12s nucleic acid detection technology.

[0030] Figure 4 .Shows the evaluation of the accuracy of multiple nucleic acid detection mediated by the RPA-CRISPR / Gs12s system. A. Evaluate the reliability of multiple nucleic acid detection technology for simulated ASFV, SVA and CSFV clinical infection samples; B. Use qPCR technology to verify the simulated clinical samples infected with the three viruses; C. Compare the accuracy of RPA-CRISPR / Gs12s and qPCR detection technologies; D. Schematic diagram of the dual nucleic acid detection technology mediated by the RPA-CRISPR / Gs12s system; E. Naked eye visualization detection of ASFV and SVA virus nucleic acids mediated by RPA-CRISPR / Gs12s technology in simulated clinical samples. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0032] Example 1. Evaluation of the base preference of Gs12s nuclease for different ssDNA-FQ reporter genes

[0033] Using bioinformatics strategies, the inventors discovered a batch of candidate Cas12a family homologous proteins Gs12 from metagenomic data. In view of the trans-cutting activity of Gs12 nuclease on dsDNA, in order to explore the base preference of these candidate Gs12 nucleases for ssDNA-FQ reporter genes, this embodiment systematically evaluates the base preference of 21 candidate Cas12a family homologous proteins for different sDNA-FQ reporter genes. The bases of ssDNA-FQ reporter genes are TATAT, TTTTT, GGGGG, etc., and the modified fluorescent group is ROX (Table 1).

[0034] Secondly, in this embodiment, the target dsDNA is selected as the African swine fever P72 gene, and the PAM is TTTA, and its sequence is: 5′-CTGTAACGCAGCACAGCTGAACCGTTCTGAAGAAGAAGAAAGTTAA TAGCAGATGCCGATACCACAAGATCAGCCGTAGTGATAGACCCCACGTAATCCGTGTCCCAACTAATATAAAATTCTCTTGCTCTGGATACGTTAATATGACCACTGGGTTGGTATTCCTCCCGTGGCTTCAAAGCAAAGGTAATCATCATCGCACCCGGATCATCGGGGGTTTTAATCGCATTGCCTCCGTAGTGGAAGGGTATGTAAGAGCTGCAGAACTTTGATGGAAATTTATCGATAAGATTGATACCATG AGCAGTTACGGAAATGTTTTTAATAATAGGTAATGTGATCGGATACGTAACGGGGCTAATATCAGATATAGATGAACATGCGTCTGGAAGAGCTGTATCTCTATCCTGAAAGCTTATCTCTGCGTGGT GAGTGGGCTGCATAATGGCGTTAACAACATGTCCGAACTTGTGCCAATCTCGGTGTTGATGAGGATTTTGATCGGAGATGTTCCAGGTAGGTTTTAATCCTATAAACATATATTCAATGGGCCATTTA AGAGCAGACATTAGTTTTTCATCGTGGTGGTTATTGTTGGTGTGGGTCACCTGCGTTTTATGGACACGTATCAGCGAAAAGCGAACGCGTTTTACAAAAAGGTTGTGTATTTCAGGGGTTACAAACAGGTTATTGATGTAAAGTTCATTATTCGTGAGCGAGATTTCATTAATGACTCCTGGGATAAACCATGG-3′; the bold mark is PAM, and the underline is the targeting sequence. The crRNA sequence is: 5′-AAUUUCUACUAUUGUAGAUU AGAGCAGACAUUAGUU UUUC -3′ (the underlined area is the targeting area). Using the pmd-18t-p72 plasmid as a template, p72-F: 5′-CTGTAACGCAGCACAGCTGA-3′, p72-R: 5′-CCATGGTTTATCCCAGGAGT-3′ as primers for PCR amplification, the P72 double-stranded DNA detection template was prepared. After E. coli codon optimization, 21 Gs12 nucleases (amino acid sequences are shown in SEQ ID NO.1-22) were synthesized, and NLS nuclear localization signals were added to their C-termini. Subsequently, they were connected to the pET-28a prokaryotic expression vector, transformed into the E. coli BL21 strain, and IPTG-induced expression was performed after positive clones were identified, and the target protein was obtained by affinity chromatography purification.

[0035] Next, the trans-cleavage activity of Gs12 nuclease was evaluated, mainly using the following reaction system: 10×CutSmartBuffer 2μL, 250nM Gs12-NLS or LbCas12a nuclease, 500nM crRNA, 50nM P72 target amplification product, 400nM ssDNA-FQ reporter gene (ROX-random-BHQ2 / ROX-N5-BHQ2, Table 1). The reaction was carried out at 37°C for 15min, and then the reaction was terminated at 98°C for 2min. The negative control was set to not add the target gene template, that is, no p72 gene amplification product was added. After the reaction was completed, 80μL DEPC water was added to the reaction solution, and the fluorescence value was read with a multifunctional microplate reader, where ROX was modified to excitation light 576nm and emission light 601nm.

[0036] Table 1. Base sequences of 8 ssDNA-FQ reporter genes

[0037]

[0038]

[0039] The results are as follows Figure 1As shown in A and 1B, the candidate gene editing enzyme Gs12 in this embodiment was independently discovered from the metagenome by the inventor using a bioinformatics strategy (the sequence is shown in SEQ ID NO.1-21). Through comparative studies, it was found that except for Gs12-21, the 20 candidate Gs12s nucleases all had good trans-cleavage activity against the ssDNA-FQ reporter gene with a random base sequence ( Figure 1 C). Among them, Gs12-1 has a preference for ssDNA-FQ with base "AAAAA", Gs12-4, Gs12-10 and LbCas12a have a preference for ssDNA-FQ with base "TTTTT". Gs12-9 has a preference for ssDNA-FQ reporter gene with base "TATAT". Gs12-18 has a preference for ssDNA-FQ reporter gene with base "TTATT". It is preliminarily speculated that different combinations of the four nucleases Gs12-1, Gs12-4, Gs12-9 and Gs12-18 can be used for multiple nucleic acid detection technology mediated by the CRISPR-Gs12 system.

[0040] Example 2. Identification of crRNA variants that can improve the specificity of multiple nucleic acid detection mediated by the CRISPR-Gs12 system

[0041] This example verifies the four Gs12 nucleases Gs12-1, Gs12-4, Gs12-9 and Gs12-18 screened above to determine whether they have ssDNA-FQ reporter gene base preference. Furthermore, this example modifies the scaffolds of their respective corresponding crRNAs in order to improve the specificity of multiple nucleic acid detection mediated by the CRISPR-Gs12 system. Combined with the secondary structural characteristics of the scaffold in crRNA, 60 different engineered crRNAs were designed for the loop region and left stem, including extension and shortening of the loop region and truncation of different left stem lengths. Finally, the candidate crRNA is matched with the above-mentioned Gs12 nuclease to evaluate its trans-cleavage activity and specificity.

[0042] In this example, the target dsDNA is the African swine fever P72 gene, and the PAM is TTTA. Its sequence: 5′-CTGTAACGCAGCACAGCTGAACCGTTCTGAAGAAGAAGAAAGTTAA TAGCAGATGCCGATACCACAAGATCAGCCGTAGTGATAGACCCCACGTAATCCGTGTCCCAACTAATATAAAATTCTCTTGCTCTGGATACGTTAATATGACCACTGGGTTGGTATTCCTCCCGTGGCTTCAAAGCAAAGGTAATCATCATCGCACCCGGATCATCGGGGGTTTTAATCGCATTGCCTCCGTAGTGGAAGGGTATGTAAGAGCTGCAGAACTTTGATGGAAATTTATCGATAAGATTGATACCATGAGCAGTTACGGAAATGTTTTTAATAATAGGTAATGTGATCGGATACGTAACGGGGCTAATATCAGATATAGATGAACATGCGTCTGGAAGAGCTGTATCTCTATCCTGAAAGCTTATCTCTGCGTGGTGAGTGGGCTGCATAATGGCGTTAACAACATGTCCGAACTTGTGCCAATCTCGGTGTTGATGAGGATTTTGATCGGAGATGTTCCAGGTAGGTTTTAATCCTATAAACATATATTCAATGGGCCATTTA AGAGCAGACATTAGTTTTTC ATCGTGGTGGTTATTGTTGGTGTGGGTCACCTGCGTTTTATGGACACGTATCAGCGAAAAGCGAACGCGTTTTACAAAAAGGTTGTGTATTTCAGGGGTTACAAACAGGTTATTGATGTAAAGTTCATTATTCGTGAGCGAGATTTCATTAATGACTCCTGGGATAAACCATGG-3′; The bold mark is the PAM, and the underlined is the targeting sequence. The crRNA sequence is: 5′-AAUUUCUACUAUUGUAGAUU AGAGCAGACAUUAGUUUUUC-3′ (the underlined area is the targeting area). Using the pmd-18t-p72 plasmid as a template, p72-F: 5′-CTGTAACGCAGCACAGCTGA-3′ and p72-R: 5′-CCATGGTTTATCCCAGGAGT-3′ were used as primers for PCR amplification to obtain P72 double-stranded DNA. First, the crRNA designed for the P72 gene was used to detect the trans-cutting activity specificity of Gs12-1, Gs12-4, Gs12-9 and Gs12-18 and the corresponding ssDNA-FQ reporter gene. Secondly, the scaffold in the crRNA designed for the P72 gene was engineered (Table 2). In order to detect whether the different crRNA variants corresponding to the candidate scaffold have high targeted cutting specificity, the CSFV-E2 gene, SVA-VP1 gene and ASFV-P72 gene were used as targets, and the specificity of multiple nucleic acid detection was tested by setting the Gs12 nuclease and crRNA variants.

[0043] Table 2. crRNA variant sequences of engineered scaffolds

[0044]

[0045]

[0046]

[0047]

[0048] The following reaction system was used to evaluate the in vitro trans-cleavage activity: 2 μL of 10×CutSmart Buffer, 250 nM of any nuclease of Gs12-1, Gs12-4, Gs12-9 or Gs12-18, 500 nM of the above-mentioned modified crRNA, 2 μL of P72 target amplification product, and 400 nM ssDNA-FQ reporter gene (ROX-AAAAA-BHQ2, ROX-TTTTT-BHQ2, ROX-TATAT-BHQ2, ROX-TTATT-BHQ2, ROX-random-BHQ2). The reaction was carried out at 37°C for 15 min, and then terminated at 98°C for 2 min. After the reaction was completed, 80 μL of DEPC water was added to the reaction solution, and the relative fluorescence intensity was read using a multifunctional microplate reader, where ROX was modified to excitation light 576 nm and emission light 601 nm.

[0049] Figure 2A is a schematic diagram of the multiple nucleic acid detection technology based on CRISPR-Gs12 and the corresponding crRNA variants. Experiments have shown that Gs12-1, Gs12-4, Gs12-9 and Gs12-18 can specifically cut ssDNA-FQ reporter genes with bases of 'AAAAA', 'TTTTT', 'TATAT' and 'TTATT' respectively ( Figure 2 B). Through screening, it was found that the crRNA variants corresponding to scaffold-54, scaffold-20, scaffold-31 and scaffold-42 can be specifically cleaved by Gs12-1, Gs12-4, Gs12-9 and Gs12-18 nucleases, respectively, and do not interfere with each other ( Figure 2 C); further identified crRNA variants suitable for multiple nucleic acid detection mediated by the CRISPR-Gs12 system ( Figure 2 D). Subsequently, the ASFV-P72 gene, SVA-VP1 gene and CSFV-E2 gene were used as targets, and corresponding specific crRNAs were designed. It was found that the optimal combination of Gs12-1 / scaffold-54, Gs12-4 / scaffold-20, Gs12-9 / scaffold-31 and Gs12-18 / scaffold-42 could specifically detect viral targets ( Figure 2 E). Thus, the optimal combination conditions of CRISPR-Gs12 and crRNA variants were determined, thereby achieving multiple nucleic acid detection.

[0050] Example 3. Establishment of a triple detection technology of viral nucleic acid mediated by CRISPR-Gs12 and corresponding crRNA variants

[0051] This example evaluates the triple detection technology of viral nucleic acids mediated by CRISPR-Gs12 and crRNA variants, and develops a triple nucleic acid detection kit for the coding gene VP1 of Seneca virus (SVA), the coding gene E2 of classical swine fever virus (CSFV), and the coding gene P72 of African swine fever virus (ASFV).

[0052] In order to detect CRISPR cleavage signals by microplate reader to achieve triple nucleic acid detection, specific ssDNA-FQ reporter genes with non-interfering excitation wavelengths of FAM (excitation wavelengths of 492 and 517 nm), HEX (excitation wavelengths of 535 and 550 nm), and ROX modified (excitation wavelengths of 576 and 601 nm) were synthesized (Table 3); specific crRNAs were designed for E2, VP1, and P72 genes according to the corresponding crRNA variants (Table 3); and a multiple nucleic acid detection method was established by combining RPA amplification technology and CRISPR / Gs12s system. The gene sequence of CSFV-E2 is: 5′CGGGACCGTCAAGGCTATTTGCGTGGCGGGCTCCTTTAAAGTCACTG CACTCAGCGTGGTCAGCAGGAGGTACTTGGCATCATTACACAAGAGGGCTCTACCCACGTCTGTAACATTTGAGCTCTTGTTCGACGGGACCAACCCATCTACTGAGGAAATGGGAGACGACTTTGGGTTCGGGCTGTGCCCTTTCGATACTAGTCCTGTTGTCAAGGGGAAGTACAATACAACCTTATTGAACGGCAGTGCCTTCTATCTTGTCTGCCCAATAGGGT GGACGGGTGTCGTAGAGTGCACAGCGGTGAGCCCTACAACTCTGAGAACGGAAGTGGTAAAGACCTTTAGGAGAGATAAGCCTTTCGCATAGAACGGATTGCGTGACCACCACGGTGGAAAATGAGGATTTATTCTACTGTAAGTTGGGGGGCAACTGGACATGCGTCAAAGGTGAACCAGTGGTCTACACAGGGGGGCTAGTAAGACAATGCAGATGGTGTGGAT-3′;The gene sequence of SVA-VP1 is: 5′CGTTTCGGCCTGTACGTCAATCCGTCTGACAGTGGCGTTCTCGCTAAC ACTTCACTGGATTTCAATTTTTACAGCTTGGCCTGTTTCACTTACTTTAGATCAGACCTTGAAGTCACGGTGGTCTCACTGGAGCCAGATCTGGAATTCGCTGTAGGGTGGTTCCCCTCTGGCAGTGAGTACCAGGCTTCCAGCTTTGTCTACGACCAACTGCATGTACCCTACCACTTTACTGGGCGCACTCCCCGCGCTTTCACCAGCAAGGGTGGAAAGGTATCTTTCGTGCTTCCTTGGAACTCTGTCTCATCCGTGCTTCCCGTGCGCTGGGGGGGCGCTTCCAAGCTTTCTTCCGCCACGCGGGGTCTACCGGCTCATGCTGACTGGGGGACCATTTACGCCTTCATCCCCCGTCCTAATGAGAAGAAAAGCACCGCTGTAAAGCACGTGGCGGTGTACGTTCGGTACAAGAACGCGCGTGCCTGGTGCCCCAGCATGCTCCCCTTCCGCAGCTACAAGCAGAAGATGCTGAT-3′;The gene sequence of ASFV-P72 is: 5′AGGTACTGTAACGCAGCACAGCTGAACCGTTCTGAAGAAGAAGAAA GTTAATAGCAGATGCCGATACCACAAGATCAGCCGTAGTGATAGACCCCACGTAATCCGTGTCCCAACTAATATAAAATTCTCTTGCTCTGGATACGTTAATATGACCACTGGGTTGGTATTCCTCCCGTGGCTTCAAAGCAAAGGTAATCATCATCGCACCCGGATCATCGGGGGTTTTAATCGCATTGCCTCCGTAGTGGAAGGGTATGTAAGAGCTGCAGAACTTTGATGGAAATTTATCGATAAGATTGATACCATGAGCAGTTACGGAAATGTTTTTAATAATAGGTAATGTGATCGGATACGTAACGGGGCTAATATCAGATATAGATGAACATGCGTCTGGAAGAGCTGTATCTCTATCCTGAAAGCTTATCTCTGCGTGGTGAGTGGGCTGCATAATGGCGTTAACAACATGTCCGAACTTGTGCCAATCTCGGTGTTGATGAGGATTTTGATCGGAGATGTTCCAGGTAGGTTTTAATCCTATAAACATATATTCAATGGGCCATTTAAGAGCAGACATTAGTTTTTCATCGTGGTGGTTATTGTTGGTGTGGGTCACCTGCGTTTTATGGACACGTATCAGCGAAAAGCGAACGCGTTTTACAAAAAGGTTGTGTATTTCAGGGGTTACAAACAGGTTATTGATGTAAAGTTCATTATTCGTGAGCGAGATTTCATTAATGACTCCTGGGATAAACCATGG-3′. RPA amplification primer pairs were designed for the above three genes (Table 3).;

[0053] The experimental process of multiple nucleic acid detection mediated by the multiple RPA-CRISPR / Gs12s system is as follows: first perform multiple RPA amplification, and resuspend one RPA pellet in each tube with 29.5μL rehydration buffer in the TwistAmp basic RPA kit. The total reaction volume is 25μL, and the system contains: 0.4μL RPA-E2-F / R (10μM), 0.5μL RPA-VP1-F / R (10μM) and 0.3μL RPA-P72-F / R (10μM) (Table 3), 14.75μL resuspended RPA solution, 0.6μL RNA reverse transcriptase (100U / mL) and 1.25μL MgAc (280mM) and 3μL sample template. React at 39°C for 30 minutes. When performing sensitivity testing of multiplex RPA combined with Gs12s multiplex nucleic acid detection, the sample templates were 10-fold gradient dilutions of pmd-18t-E2 / VP1 / p72 single plasmid and mixed plasmid.

[0054] After the multiple RPA amplification is completed, take 2 μL of the amplified product for the subsequent CRISPR / Gs12s nucleic acid detection system. Use the following system: 10×CutSmart Buffer 2 μL, Gs12-1, Gs12-4 and Gs12-18 nucleases 250 nM each, crRNA 500 nM each, FAM-AAAAA-BHQ1, HEX-TTTTT-BHQ1 and ROX-TTATT-BHQ2 250 nM each. The reaction was run at 37°C for 15 min and inactivated at 98°C for 2 min. Use a multifunctional blue light instrument to visualize the fluorescence reaction results with the naked eye. Or add 80 μL of DEPC water after the reaction is completed and use Fluorescence detection was performed using a microplate fluorescence reader, with excitation / emission wavelengths of 492 / 601 nm for FAM, 535 / 550 nm for HEX, and 576 / 601 nm for ROX.

[0055] Table 3. Reporter genes, crRNAs, and primer pairs in multiplex nucleic acid detection mediated by the RPA-CRISPR / Gs12s system

[0056] Name Primer Sequence (5'-3') FAM-AAAAA-BHQ1 5’-FAM / AAAAA / 3’BHQ1 HEX-TTTTT-BHQ1 5’-HEX / TTTTT / 3’BHQ1 ROX-TTATT-BHQ2 5’-ROX / TTATT / 3’BHQ2 SVA-VP1-crRNA UCUACCCCGUAGAUCAGCUUGGCCUGUUUCACUU CSFV-E2-crRNA UAAUUUCUACCCGUAGAUGAUACUAGUCCUGUUGUCAA ASFV-P72-crRNA UCUACAAAAGUAGAUAGAGCAGACAUUAGUUUUUC RPA-E2-F TCAGCGTGGTCAGCAGGAGGTACTTGGCATCAT RPA-E2-R CTACGACACCCGTCCACCCTATTGGGCAGACAA RPA-VP1-F TCTGACAGTGGCGTTCTCGCTAACACTTCACTG RPA-VP1-R TTGGTCGTAGACAAAGCTGGAAGCCTGGTACTC RPA-P72-F GCCAATCTCGGTGTTGATGAGGATTTTGATCGGAG RPA-P72-R CCATGGTTTATCCCAGGAGTCATTAATGAAATCTC p72-F AGGTACTGTAACGCAGCACAGC p72-R ATGAAATCTCGCTCACGAAT E2-F CGGGACCGTCAAGGCTATTT E2-R ATCCACACCATCTGCATTGTCT VP1-F CGTTTCGGCCTGTACGTCA VP1-R ATCAGCATCTTCTGCTTGT

[0057] The results are as follows Figure 3 As shown in Figure A, the specificity of using RPA-CRISPR / Gs12s technology to detect SVA, CSFV and ASFV was first evaluated. The results showed that Gs12-1, Gs12-4 and Gs12-18, corresponding to ssDNA-FQ reporter genes modified with FAM, HEX and ROX, and specific crRNA variants, could specifically distinguish the three viral nucleic acid targets (Figure 3 B). The detection sensitivity of the technology was further evaluated by diluting the test samples in multiples ( Figure 3 C). The results showed that the triple nucleic acid detection technology mediated by RPA-CRISPR / Gs12s technology had a detection sensitivity of single copy / microliter for the three viral nucleic acids of SVA, CSFV and ASFV ( Figure 3 D). It shows that the triple nucleic acid detection technology and kit for SVA, CSFV and ASFV have been preliminarily established.

[0058] Example 4. Evaluation of the accuracy of multiplex RPA-CRISPR / Gs12s technology in detecting different viral target genes

[0059] Further, this example evaluates the accuracy of RPA-CRISPR / Gs12s technology in detecting SVA, CSFV and ASFV. First, 28 simulated clinical samples were prepared, and then multiple RPA amplification was performed using the kit. 29.5μL rehydration buffer in the TwistAmp basicRPA kit was selected to resuspend 1 RPA precipitate pellet in each tube. The total reaction volume was 25μL, and the system contained: 0.4μL RPA-E2-F / R (10μM), 0.5μL RPA-VP1-F / R (10μM) and 0.3μL RPA-P72-F / R (10μM) (Table 3), 14.75μL resuspended RPA solution, 0.6μL RNA reverse transcriptase (100U / mL) and 1.25μL MgAc (280mM) and 3μL sample template. React at 39°C for 30 minutes.

[0060] In this example, the simulated clinical samples were prepared by mixing ASFV, SVA and CSFV viruses separately, and clinical samples infected with two or three viruses were prepared. TM Nucleic acid was extracted from all infected samples using a DNA extraction kit (Lucigen). An appropriate amount of tissue or blood was added to 80 μL of the reagent solution and reacted at 65°C for 5 minutes and at 95°C for 5 minutes to extract genomic DNA.

[0061] After the multiple RPA amplification is completed, take 2μL of the amplification product for the subsequent multiple Gs12s nucleic acid detection system. Use the following system: 10×CutSmart Buffer 2μL, Gs12-1, Gs12-4 and Gs12-18 nucleases 250nM each, specific crRNA 500nM each, 3μL ssDNA-FQ reporter gene (FAM-AAAAA-BHQ1, HEX-TTTTT-BHQ1 and ROX-TTATT-BHQ2 250nM each). The reaction is run at 37°C for 15min and inactivated at 98°C for 2min. Use a multifunctional blue light instrument to visualize the fluorescence reaction results with the naked eye. Or add 80μL DEPC water after the reaction is completed and use Fluorescence detection was performed using a microplate fluorescence reader, with excitation / emission wavelengths of 492 / 601 nm for FAM, 535 / 550 nm for HEX, and 576 / 601 nm for ROX.

[0062] In the double-plex (SVA and ASFV) visual nucleic acid detection, double-plex RPA amplification was first performed, and 29.5μL rehydration buffer in the TwistAmpbasic RPA kit was selected to resuspend one RPA pellet in each tube. The total reaction volume was 25μL, and the system contained: 0.5μL RPA-VP1-F / R (10μM) and 0.3μL RPA-P72-F / R (10μM), 14.75μL resuspended RPA solution, 0.6μL RNA reverse transcriptase (100U / mL) and 1.25μL MgAc (280mM) and 3μL sample template. The reaction was carried out at 39°C for 30 minutes. The double Gs12 nucleic acid detection system includes 250nM of Gs12-1 and Gs12-18 nucleases, 500nM of their corresponding crRNAs, 250nM of ROX-TTATT-BHQ2 reporter genes and FAM-AAAAA-BHQ1 reporter genes, 2μL of 10×CutSmart buffer, 2μL of multiple RPA amplification products, and 20μL of the total reaction system. The reaction temperature is 37℃ for 15 minutes and 98℃ for 2 minutes. Finally, the PCR tube containing 20μL of CRISPR / Gs12s reactants was placed on the blue light detector and photographed by a smartphone.

[0063] Comparison of the accuracy of simulated clinical samples with qPCR / RT-qPCR, using SYBR High-Sensitivity qPCR SuperMix (Novoprotein) was used to detect ASFV, CSFV, and SVA by qPCR / RT-qPCR. The reaction system was: 10 μL 2×SYBR mix, 0.4 μL qPCR-P72-F / R or qPCR-E2-F / R or qPCR-VP1-F / R primers (Table 4), and 2 μL of genomic DNA from simulated clinical samples.

[0064] Table 4. Primer pairs for qPCR and RT-qPCR amplification

[0065] qPCR-P72-F CTGCTCATGGTATCAATCTTATCGA qPCR-P72-R GATACCACAAGATCAGCCGTA qPCR-E2-F TGAGGAAATGGGAGACGA qPCR-E2-R CACCCTATTGGGCAGACA qPCR-VP1-F AGCAAGGGTGGAAAGGTA qPCR-VP1-R GACCATTTACGCCTTCATC

[0066] The reaction conditions were as follows: 95°C for 60 seconds; followed by 35 cycles of 95°C for 20 seconds and 60°C for 45 seconds; and finally 40°C for 5 minutes. Fluorescence information was collected during the 60°C annealing step of each cycle. For the SVA assay, additional Luna was added at 55°C before the start of the procedure. RT enzyme mixture (NEB) was reacted for 20 minutes to perform the reverse transcription process, and the rest of the procedures were the same as above. All detections were performed using CFX real-time fluorescence quantitative PCR detection (Bio-Rad). The qPCR reaction results were compared with the results of the established multiplex nucleic acid detection for accuracy evaluation.

[0067] The results are as follows Figure 4 As shown in A, a total of 28 crude nucleic acid extracts were prepared as simulated clinical SVA, CSFV and ASFV infection samples. To ensure the reliability of the test, each sample was tested three times. Through multiple RPA-CRISPR / Gs12s technology detection, it was found that 6 samples were ASFV positive, 4 samples were SVA positive, 2 samples were CSFV positive, 2 samples were ASFV and SVA co-infection positive, 1 sample was ASFV and CSFV co-infection positive, 1 sample was SVA and CSFV co-infection positive, 1 sample was ASFV, SVA and CSFV co-infection positive, 1 sample was ASFV, SVA and CSFV co-infection positive, 11 samples were negative ( Figure 4 A). The results are completely consistent with those detected by qPCR or RT-qPCR ( Figure 4 B, C).

[0068] In addition, this embodiment also establishes a new technology based on the RPA-CRISPR / Gs12s system to achieve dual visualization detection of viral nucleic acids ( Figure 4D). The results showed that this technology can be used to visualize the detection of ASFV-positive samples (samples 1 and 11), SVA-positive samples (samples 16, 20 and 23), ASFV and SVA co-positive samples (sample 3) and negative samples (samples 4, 9 and 10). It can be seen with the naked eye that when the sample is only ASFV positive, the solution reaction appears red, which is the fluorescence excited by ROX; when the sample is only SVA positive, the solution reaction appears green, which is the fluorescence excited by FAM; when the sample is ASFV and SVA co-positive, the solution reaction appears yellow ( Figure 4 E). It can be seen that the present invention has successfully established a multiplex nucleic acid detection technology mediated by a multiplex RPA-CRISPR / Gs12s system, and developed triple and double virus detection kits for SVA, CSFV and ASFV.

[0069] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technician familiar with the field can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A multiple nucleic acid detection kit based on CRISPR / Gs12 system, characterized in that: include: Gs12 nuclease, the Gs12 nuclease includes at least two of Gs12-1, Gs12-4, Gs12-9 and Gs12-18 nucleases, the amino acid sequence of Gs12-1 is shown in SEQ ID NO.1, the amino acid sequence of Gs12-4 is shown in SEQ ID NO.4, the amino acid sequence of Gs12-9 is shown in SEQ ID NO.9, and the amino acid sequence of Gs12-18 is shown in SEQ ID NO.18; The reporter gene specifically corresponding to the Gs12 nuclease, the reporter gene sequence corresponding to the Gs12-1 nuclease is 5´-AAAAA-3´, the reporter gene sequence corresponding to the Gs12-4 nuclease is 5´-TTTTT-3´, the reporter gene sequence corresponding to the Gs12-9 nuclease is 5´-TATAT-3´, and the reporter gene sequence corresponding to the Gs12-18 nuclease is 5´-TTATT-3´, and the 5´ end of the reporter gene is modified with a fluorescent group whose excitation wavelengths do not interfere with each other, and the 3´ end is modified with a quenching group; A cRNA corresponding to the Gs12 nuclease, wherein the cRNA contains a scaffold and a targeting sequence.

2. The kit according to claim 1, characterized in that The scaffold sequence in the crRNA of the Gs12-1 nuclease is shown as SEQ ID NO.22, the scaffold sequence in the crRNA of the Gs12-4 nuclease is shown as SEQ ID NO.23, the scaffold sequence in the crRNA of the Gs12-9 nuclease is shown as SEQ ID NO.24, and the scaffold sequence in the crRNA of the Gs12-18 nuclease is shown as SEQ ID NO.

25.

3. A kit for multiple detection of Seneca virus, classical swine fever virus and African swine fever virus mediated by CRISPR / Gs12 system, characterized in that: include: Gs12 nuclease, wherein the Gs12 nuclease is selected from any three of Gs12-1, Gs12-4, Gs12-9 and Gs12-18 nucleases, wherein the amino acid sequence of Gs12-1 is shown in SEQ ID NO.1, the amino acid sequence of Gs12-4 is shown in SEQ ID NO.4, the amino acid sequence of Gs12-9 is shown in SEQ ID NO.9, and the amino acid sequence of Gs12-18 is shown in SEQ ID NO.18; The reporter gene specifically corresponding to the Gs12 nuclease, the reporter gene sequence corresponding to the Gs12-1 protein is 5´-AAAAA-3´, the reporter gene sequence corresponding to the Gs12-4 protein is 5´-TTTTT-3´, the reporter gene sequence corresponding to the Gs12-9 protein is 5´-TATAT-3´, and the reporter gene sequence corresponding to the Gs12-18 protein is 5´-TTATT-3´, and the 5´ end of the reporter gene is modified with a fluorescent group whose excitation wavelengths do not interfere with each other, and the 3´ end is modified with a quenching group; The cRNA specifically targets the Seneca virus VP1 gene, the classical swine fever virus E2 gene and the African swine fever virus P72 gene. The cRNA sequence targeting the Seneca virus VP1 gene is shown in SEQ ID NO.26, the cRNA sequence targeting the classical swine fever virus E2 gene is shown in SEQ ID NO.27, and the cRNA sequence targeting the African swine fever virus P72 gene is shown in SEQ ID NO.

28.

4. The kit according to claim 3, characterized in that Also included are primers for amplifying the Seneca virus VP1 gene, the classical swine fever virus E2 gene, and the African swine fever virus P72 gene.

5. The kit according to claim 4, characterized in that The RPA primer sequence for amplifying the Seneca virus VP1 gene is shown in SEQ ID NOs. 29 and 30, the RPA primer sequence for amplifying the classical swine fever virus E2 gene is shown in SEQ ID NOs. 31 and 32, and the RPA primer sequence for amplifying the African swine fever virus P72 gene is shown in SEQ ID NOs. 33 and 34.

6. The kit according to any one of claims 3 to 5, characterized in that The Gs12 nucleases are Gs12-1, Gs12-4 and Gs12-18 nucleases.

7. A kit for dual visualization detection of Seneca virus and African swine fever virus based on CRISPR / Gs12 system, characterized in that: include: Gs12-1 and Gs12-18 nucleases, wherein the amino acid sequence of Gs12-1 is shown in SEQ ID NO.1, and the amino acid sequence of Gs12-18 is shown in SEQ ID NO.18; The reporter gene sequence corresponding to the Gs12-1 nuclease is 5´-AAAAA-3´, and the reporter gene sequence corresponding to the Gs12-18 nuclease is 5´-TTATT-3´, and the 5´ end of the reporter gene is modified with a fluorescent group FAM or ROX, and the 3´ end is modified with a quenching group; The cRNA sequence specifically targeting the Seneca virus VP1 gene is shown in SEQ ID NO.26, and the cRNA sequence specifically targeting the African swine fever virus P72 gene is shown in SEQ ID NO.

28.

8. The kit according to claim 7, characterized in that Also included are primers for amplifying the Seneca virus VP1 gene and the African swine fever virus P72 gene.

9. The kit according to claim 8, characterized in that The RPA primer sequences for amplifying the Seneca virus VP1 gene are shown in SEQ ID NOs. 29 and 30, and the RPA primer sequences for amplifying the African swine fever virus P72 gene are shown in SEQ ID NOs. 33 and 34.

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