Probe, primer and kit for identifying gene type I, gene type II and gene I / II recombinant African swine fever viruses

Abstract

The invention relates to a probe, a primer and a kit for identifying gene I type, gene II type and gene I / II recombinant African swine fever viruses. Probes for identifying gene I type, gene II type and gene I / II recombinant African swine fever viruses comprise SNP (Single Nucleotide Polymorphism) typing probes P1 and P2 aiming at gene I type and gene II type ASFV B646L genes and a specific probe P3 aiming at gene II type ASFV CP204L genes, and nucleotide sequences of the P1, the P2 and the P3 are as shown in SEQ ID NO.5-7. The application not only can effectively distinguish the gene I type ASFV from the gene II type ASFV, but also can identify the gene I / II type recombinant African swine fever virus currently popular in China.

Description

Technical Field

[0001] The present application relates to the field of virus detection technology, and specifically to probes, primers and kits for identifying genotype I, genotype II and genotype I / II recombinant African swine fever viruses. Background Art

[0002] African swine fever (ASF) is caused by the African swine fever virus (ASFV), an acute, highly contagious disease characterized by fever and hemorrhage, primarily affecting domestic pigs and wild boars. Based on differences in the 478bp nucleotide sequence at the 3' end of the ASFV B646L gene, ASFV is divided into 24 genotypes. Since the introduction of genotype IIASFV into my country in 2018, it has caused significant losses to the country's pig farming industry. In 2021, Sun et al. reported the emergence of a weak genotype I ASFV strain in China. In 2023, Zhao et al. reported the emergence of a virulent genotype I / II recombinant ASFV strain in my country. Furthermore, the gene-deficient ASFV had no protective effect against this virulent strain, further increasing the difficulty of ASF prevention and control in my country.

[0003] Except for the African continent, ASFV genotype I and genotype II are mainly prevalent in other countries and regions. Currently, ASFV genotyping and identification are also focused on these two genotypes. The commonly used methods are SNP typing and identification of the ASFV B646L gene and typing and identification based on other genes (such as E296R gene, CP204L gene, E183L gene, etc.). However, analysis of the reported genotype I / II recombinant ASFV virulent strain genome sequence shows that this strain is different from the previously reported ASFV. Although its B646L gene belongs to genotype I according to the traditional typing method, the EP402L, E296R, CP204L, E183L and other gene sequences are completely consistent with the type II ASFV gene sequences since 2018. Obviously, some of the methods established in the early stage to distinguish between genotype I and type II ASFV are not suitable for the genotyping and identification of the current ASFV prevalent strains in my country.

[0004] Fluorescence PCR is a commonly used nucleic acid detection technology and one of the diagnostic methods recommended by WOAH. It has high sensitivity and specificity and can quickly detect viral nucleic acids under laboratory conditions. Therefore, the use of fluorescence PCR to identify the existing genotype I, type II, and I / II recombinant ASFV in China has practical significance and can provide technical support for the rapid identification of ASFV, epidemic investigation, and virus traceability analysis. Summary of the Invention

[0005] In order to better identify genotype I, type II, and I / II recombinant ASFV, the present application provides probes, primers, and kits for identifying genotype I, genotype II, and genotype I / II recombinant African swine fever viruses. Primers targeting the 3' end of the ASFV B646L gene and specific probes P1 and P2 specific to type I and type II, respectively labeled with FAM and VIC fluorescein, which can perform SNP typing, as well as a pair of primers for detecting the genotype II ASFV CP204L gene and a specific probe P3 labeled with ROX fluorescein are designed, and a kit is assembled on this basis.

[0006] The present application provides probes for identifying genotype I, genotype II and genotype I / II recombinant African swine fever viruses, including SNP typing probes P1 and P2 for the genotype I and genotype II ASFV B646L genes, and a specific probe P3 for the genotype II ASFV CP204L gene. The nucleotide sequences of P1, P2 and P3 are shown in SEQ ID NO.5-7.

[0007] Preferably, the 5' end of P1 is labeled with FAM, the 3' end is labeled with MGB, and the oligonucleotide G at position 7 is a locked nucleotide.

[0008] Preferably, the 5' end of P2 is labeled with VIC, the 3' end is labeled with MGB, and the oligonucleotide A at position 7 is a locked nucleotide.

[0009] Preferably, the 5' end of P3 is labeled with ROX, and the 3' end is labeled with MGB.

[0010] The present application also provides primers for identifying genotype I, genotype II and genotype I / II recombinant African swine fever viruses. The nucleotide sequence of the primer for detecting the ASFV B646L gene in the above-mentioned probe is shown in SEQ ID NO.1-2.

[0011] Preferably, the nucleotide sequence of the primer for detecting the genotype II ASFV CP204L gene is as shown in SEQ ID NO.3-4.

[0012] The present application also provides the use of any of the above-mentioned probes or the primers described in claim 5 or 6 in the preparation of a kit for detecting African swine fever virus.

[0013] The present application also provides a kit for identifying genotype I, genotype II and genotype I / II recombinant African swine fever virus, comprising a reaction solution, DNA polymerase, a positive control, a negative control and nuclease-free sterile deionized water, wherein the reaction solution includes any of the above-mentioned probes or the above-mentioned primers.

[0014] The present application also provides a method for identifying genotype I, genotype II and genotype I / II recombinant African swine fever virus. The above-mentioned kit is used for PCR detection, and the type of African swine fever virus strain is determined according to the fluorescence signal results. If only the FAM channel is detected as positive, it is genotype I ASFV; if both the VIC and ROX channels are detected as positive, it is genotype II ASFV; if both the FAM and ROX channels are detected as positive, it is genotype I / II recombinant ASFV.

[0015] Preferably, the reaction system for each reaction is: 2×12.5 μL of the reaction solution, 9.75 μL of the nuclease-free sterile deionized water, 2.5 μL of the template, and 0.25 μL of the DNA polymerase. The reaction procedure is: pre-denaturation at 94°C for 20 s; denaturation at 98°C for 5 s, annealing and extension at 59°C for 30 s, and fluorescence collection, for a total of 45 cycles.

[0016] The beneficial effects of this application are as follows:

[0017] Since ASF entered my country in 2018, the isolated ASFV strains include genotype II virulent strains, genotype I weak strains, and genotype I / II recombinant virulent strains. Although there are many ASFV detection methods, there are few reports on the one-time identification of specific ASFV genotypes (especially recombinant types). This application uses the SNP genotyping method to distinguish genotype I and genotype II ASFV, combined with the probe method to identify the genotype II ASFV CP204L gene, to develop a multiplex fluorescent PCR kit for identifying genotype I, genotype II, and genotype I / II recombinant African swine fever virus. The kit has high sensitivity, good specificity and repeatability, and high detection accuracy. It can provide an effective diagnostic tool for rapid typing, monitoring, and tracing of ASFV in the future, and is of great significance to the epidemiological investigation and prevention and control of ASF in my country.

[0018] The probes and primers of the present application have high specificity, good reproducibility and high sensitivity.

[0019] The method of the present application for identifying genotype I, genotype II and genotype I / II recombinant African swine fever virus is not only highly accurate but also has a quick identification process. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparison of the gene-specific site sequences of genotype I, type II, and I / II recombinant ASFVB646L.

[0021] Figure 2 Comparison of the specific site sequences of the CP204L gene of genotype I, type II, and recombinant I / II ASFV.

[0022] Figure 3Enzyme digestion analysis of recombinant plasmids pUC19-p72Ⅰ and pUC19-p72Ⅱ. Figure: M.DL2000 DNA Marker; 1, 2. Recombinant plasmid pUC19-p72Ⅰ; 3, 4. Recombinant plasmid pUC19-p72Ⅱ.

[0023] Figure 4 PCR identification of the recombinant plasmid pBM18A-p30Ⅱ. In the figure, M.DL2000 DNA Marker; 1-6. PCR amplification of the p30Ⅱ gene fragment in bacterial culture.

[0024] Figure 5 This is the sensitivity test result of multiplex fluorescence PCR based on plasmid pUC19-p72Ⅰ.

[0025] Figure 6 This is the sensitivity test result of multiplex fluorescence PCR based on plasmid pUC19-p72ⅠI.

[0026] Figure 7 This is the sensitivity test result of multiplex fluorescence PCR based on plasmid pBM18A-p30Ⅱ.

[0027] Figure 8 In the figure, 1. Standard curve based on plasmid pUC19-p72Ⅰ; 2. Standard curve based on plasmid pUC19-p72Ⅰ; 3. Standard curve based on plasmid pBM18A-p30Ⅱ.

[0028] Figure 9 This is the specific detection result of multiplex fluorescence PCR. DETAILED DESCRIPTION

[0029] The following examples are used to illustrate the present application but are not intended to limit the scope of the present application. The experimental methods in the following examples are all conventional methods unless otherwise specified. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or in accordance with the product instructions are used. Where the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0030] A kit for identifying genotype I, genotype II, and genotype I / II recombinant African swine fever virus, comprising a 10× reaction solution, dNTPs (dATP, dTTP, dCTP, dGTP), DNA polymerase, a positive control, a negative control, and nuclease-free sterile deionized water; the reaction solution comprises any of the above-mentioned probes or primers.

[0031] To design primers and probes for identifying the ASFV B646L gene, representative genotype I, II, and I / II recombinant ASFV B646L gene sequences were selected from Genbank. Combined with the B646L genotyping method, MEGA7 software was used for comparative analysis to determine the target sequence for distinguishing between genotypes I and II. The nucleotide sequence from nt1623 to 1715 was selected as the qPCR target sequence, with nt1656G being used as the target for SNP genotyping. When designing primers and probes to identify gene I / II recombinant ASFV, SnapGene 4.36 software was first used to compare the known gene I / II recombinant ASFVNJS23-1 (PP478517), NJS23-2 (PP478518), Pig / Henan / 123014 / 2022 (OQ504954), Pig / InnerMongolia / DQDM / 2022 (OQ504955), and Pig / Jiangsu / LG / 2021 (OQ504956) genome sequences in Genbank. The results showed that the above ASFV genome sequences were not much different. Using ASFV NJS23-1 as the research object, the strains and their genotypes with identical gene sequences were compared. The results showed that the CP204L gene of the I / II recombinant ASFV was completely identical to the CP204L gene of genotype II ASFV, but had certain nucleotide differences with the CP204L gene of genotype I ASFV. Combined with the identification method of genotype I ASFV, the CP204L gene was selected as the target gene for identification of I / II recombinant ASFV. The typical genotype I, type II, and I / II recombinant ASFV CP204L genes were selected and analyzed using MEGA7 software. The nucleotide sequence of nt264-354 was determined as the detection target sequence, which contains three mutation sites that distinguish type I from I / II recombinant ASFV. During multiplex fluorescence PCR testing, if only the FAM channel tests positive, it is genotype I ASFV; if both the VIC / ROX channels test positive, it is genotype II ASFV; if both the FAM / ROX channels test positive, it is genotype I / II recombinant ASFV.

[0032] A method for identifying genotype I, genotype II and genotype I / II recombinant African swine fever virus for non-diagnostic purposes, comprising the following steps: using the above-mentioned kit for PCR detection, using genotype I ASFV YNCX-2021-060 strain and genotype II ASFV JS strain nucleic acid templates, and performing PCR amplification with primers B646LF (SEQ ID NO.8) and B646LR (SEQ ID NO.9); after digesting the PCR products with EcoR I and BamH I restriction endonucleases, the PCR products were inserted into the corresponding restriction sites of the pUC19 plasmid to obtain recombinant plasmids pUC19-p72Ⅰ and pUC19-p72; using genotype II ASFV JS strain nucleic acid templates, and performing PCR amplification with primers CP204LF (SEQ ID NO.10) and CP204LR (SEQ ID NO.11); the PCR products were directly cloned into the pBM18A vector to obtain the recombinant plasmid pBM18A-p30Ⅱ.

[0033] Using the recombinant plasmid constructed above as a template, the concentrations of primers and probes in the multiplex fluorescent PCR reaction, as well as the annealing temperature, were optimized. The optimal reaction system ultimately determined was: 2.5 μL of 10× PCR buffer, 2 μL of 2.5 mM dNTP mix, 0.4 μM each of p72 F / p72 R primers, 0.1 μM of P1, 0.1 μM of P2, 0.4 μM each of p30 F / p30 R primers, 0.4 μM of P3, 0.4 μM of Takara Taq HS, 2.5 μL of the test sample, and water was added to 25 μL. The optimal reaction procedure was: initial denaturation at 94°C for 20 s; denaturation at 98°C for 5 s; annealing and extension at 59°C for 30 s, with fluorescence collection, for a total of 45 cycles.

[0034] The above reaction system and reaction procedure were used to detect the gradient dilution of standard plasmids by multiplex fluorescence PCR. The results showed that the detection limit of this method for plasmid pUC19-p72Ⅰ was 10 2 copies / μL, and the detection limit for pUC19-p72Ⅱ was 10 2 copies / μL, and the detection limit for pBM18A-p30Ⅱ was 10 1 The logarithm of the plasmid standard copy number was used as the horizontal axis and the CT value was used as the vertical axis to draw a standard curve, and the correlation coefficient R2 was calculated. The result R2 was ≥0.9968, indicating that the template concentration and the CT value in the multiplex fluorescence PCR reaction had a good correlation.

[0035] Furthermore, the repeatability and specificity of the multiplex fluorescence PCR were measured. The test results showed that the intra-batch coefficient of variation of the CT value of the multiplex fluorescence PCR detection was 0.261%-2.921%, and the inter-batch coefficient of variation was 0.976%-3.961%, both less than 5%. The above results indicate that the established multiplex qPCR has good repeatability; the multiplex fluorescence PCR was used to detect known PCV-2, PRRSV, PPV, FMDV, PRV, CSFV, and PEDV nucleic acids. At the same time, positive controls of ASFVYNCX-2021-060, ASFV JS, and ASFVNJS23-1 nucleic acids and negative controls of healthy pig tissue DNA were set up. The results showed that only the positive control showed a specific amplification curve, and no specific amplification curve appeared in the other groups, indicating that the multiplex fluorescence PCR has good detection specificity.

[0036] Based on the above, the components were assembled into a kit, wherein each kit (50 reactions) contained 2× reaction solution (including 125 μL of 10× PCR Buffer, 100 μL of 2.5 mM dNTP Mixture, 50 μL of 0.4 μM primers p72 F, p72R, p30 F, and p30 R, 12.5 μL of 0.1 μM probes P1 and P2, 50 μL of 0.1 μM probe P3, and 125 μL of sterile deionized water without nuclease), 625 μL of TakaraTaq HS (5 U / μL), 12.5 μL of positive control (10 7 126 μL of plasmids pUC19-p72Ⅰ, pUC19-p72, pBM18A-p30Ⅱ (42 μL each) and negative control (10 7 125 μL of plasmid pUC19 (with a concentration of 10 copies / μL) and 500 μL of sterile, nuclease-free deionized water were added. The reaction system for each reaction consisted of 12.5 μL of 2× reaction solution, 9.75 μL of sterile, nuclease-free deionized water, 2.5 μL of template, and 0.25 μL of TakaraTaq HS. The reaction procedure was as follows: 94°C pre-denaturation for 20 seconds, 98°C denaturation for 5 seconds, 59°C annealing and extension for 30 seconds, and fluorescence collection for a total of 45 cycles.

[0037] The type of African swine fever virus strain was determined according to the fluorescence signal results. If only the FAM channel was detected as positive, it was genotype I ASFV; if both the VIC and ROX channels were detected as positive, it was genotype II ASFV; if both the FAM and ROX channels were detected as positive, it was genotype I / II recombinant ASFV.

[0038] The above kit was used to perform multiplex fluorescence PCR on 144 samples. The results showed that the three known genotype I / II recombinant ASFV nucleic acid samples all tested positive for recombinant ASFV I / II; the ASFV YNCX-2021-060 nucleic acid test result was positive for genotype I ASFV; the nine known genotype II ASFV nucleic acid samples all tested positive for genotype II ASFV; and all 132 ASFV-negative clinical samples tested negative. This indicates that the established multiplex fluorescence PCR has a high accuracy rate for the detection of clinical samples and can be used for the differential diagnosis of different ASFV genotypes in my country.

[0039] Sources of biological materials for this application:

[0040] The pUC19 plasmid, nucleic acids of genotype II ASFV JS (OR180113) and recombinant ASFV NJS23-1 (OR180113), ASFV-negative clinical samples, porcine circovirus type II (PCV-2), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), foot-and-mouth disease virus (FMDV-O), pseudorabies virus (PRV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), and porcine serum and tissue samples collected in 2017 are stored in this laboratory.

[0041] The pBM18A vector was purchased from Biomed Biotech Co., Ltd.;

[0042] 10× PCR Buffer, dNTP Mixture (2.5 mM each), TakaraTaq HS (5 U / μL), and DH5α competent cells were purchased from Dalian Takara Company;

[0043] Genotype I ASFVYNCX-2021-060 nucleic acid was provided by the African Swine Fever Reference Laboratory of the National Center for Exotic Diseases;

[0044] The positive nucleic acid of genotype II ASFV was provided by the external testing service platform of the School of Veterinary Medicine of Yangzhou University.

[0045] Example 1 Design and synthesis of primers and probes

[0046] Typical genotype I, II, and I / II recombinant ASFV B646L gene sequences were selected from Genbank. Combined with the B646L genotyping method, MEGA7 software was used for comparative analysis, and the nucleotide sequence of nt1623-1715 was selected as the target sequence for distinguishing genotype I and genotype II ASFV. Among them, nt1656G was used as the target site for SNP genotyping ( Figure 1 ), design the following primers and probes:

[0047] p72 F: 5'-CGTAACTGCTCATGGTATCAATCTT-3' (SEQ ID NO. 1),

[0048] p72 R: 5'-TTAATTGCATTGCCTCCGTAGT-3' (SEQ ID NO. 2),

[0049] P1: 5'-CGATAAGTTTCCA-3' (SEQ ID NO. 5), 5' end labeled with FAM, 3' end labeled with MGB, italic G is labeled with LNA,

[0050] P2: 5'-CGATAAATTTCCA-3' (SEQ ID NO. 6), 5'-end labeled with VIC, 3'-end labeled with MGB, italic A is labeled with LNA.

[0051] SnapGene 4.36 software was used to compare the known gene I / II recombinant ASFV NJS23-1 (PP478517), NJS23-2 (PP478518), Pig / Henan / 123014 / 2022 (OQ504954), Pig / InnerMongolia / DQDM / 2022 (OQ504955), and Pig / Jiangsu / LG / 2021 (OQ504956) genome sequences in Genbank. The results showed that the above ASFV genome sequences were not much different. Using ASFVNJS23-1 as the research object, Blast compared each strain with the same gene sequence and its genotype. The results showed that the CP204L gene of the I / II recombinant ASFV was completely consistent with the CP204L gene of the genotype II ASFV, but there were certain nucleotide differences with the CP204L gene of the genotype I ASFV. The CP204L gene was used as the target gene for identification of the I / II recombinant ASFV. The typical genotype I, type II, and I / II recombinant ASFV CP204L genes were selected and analyzed using MEGA7 software, and the nucleotide sequence of nt264-354 was determined as the detection target sequence ( Figure 2 ), the following primers and probes were designed (wherein the probe contained three mutation sites that distinguish type I from type I / II recombinant types):

[0052] p30 F: 5'-TGTGCTGTTTGAAGAGGAGACG-3' (SEQ ID NO.3),

[0053] p30 R: 5'-GGAGGATGTGCATTCATTGGT-3' (SEQ ID NO.4),

[0054] P3: 5'-CTTCGGAGAACATT-3' (SEQ ID NO. 7), 5'-end labeled with ROX, 3'-end labeled with MGB.

[0055] The above primers and probes were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0056] Example 2 Construction of standard plasmid

[0057] Based on the target genes screened in Example 1, specific primers for amplifying genotype I and genotype II ASFV B646L genes, as well as genotype II ASFV CP204L gene, were designed:

[0058] B646L F: 5'-CGGAATTCGGATAGAGATACAGCTCTTCCAG-3' (SEQ ID NO.8),

[0059] B646L R: 5'-CGGGATCCACTGTAACGCAGCACAGCTG-3' (SEQ ID NO.9), CP204L F: 5'-CCATGAGTCTTACCACCTC-3' (SEQ ID NO.10),

[0060] CP204L R: 5'-GGAGGTCATCTTCAAAACGG-3' (SEQ ID NO. 11).

[0061] The primers were synthesized by Qingke Biotechnology Co., Ltd.

[0062] Using nucleic acid templates of genotype I ASFV YNCX-2021-060 and genotype II ASFV JS, B646L F / B646L R were used as primers to amplify p72Ⅰ and p72Ⅱ gene fragments of 425 bp in size. After restriction endonuclease digestion, the fragments were inserted into the corresponding restriction sites of the pUC19 vector to construct recombinant plasmids. Double enzyme digestion was performed with EcoRⅠ and BamHⅠ restriction endonucleases to identify the target bands of the expected size, indicating that the recombinant plasmids were successfully constructed and named pUC19-p72Ⅰ and pUC19-p72Ⅱ ( Figure 3 ).

[0063] Using genotype II ASFV JS nucleic acid as a template, primers CP204LF / CP204L R were used to amplify the p30Ⅱ gene by PCR. The recovered PCR product was ligated with the vector pBM18A and transformed into competent cells. The colony was picked and inoculated into the culture medium. The bacterial solution was used as a template. The positive clones were identified by PCR. The result showed that the amplified size was 527bp band ( Figure 4), indicating that the recombinant plasmid was successfully constructed and named pBM18A-p30Ⅱ.

[0064] Example 3 Reaction system and reaction procedure optimization

[0065] 1. Determination of primer and probe concentration

[0066] Using the pUC19-p72 II plasmid as a template, qPCR was performed at different primer and probe concentrations, and the detection results (CT values) are shown in Table 1. The results showed that for the detection of genotype II ASFV p72 F / p72 R, the primer concentrations were 0.3 μM each, and the probe P2 concentrations were 0.2 μM and 0.4 μM; the primer concentrations were 0.4 μM each, and the probe P2 concentration was 0.1 μM; and the primer concentrations were 0.5 μM each, and the probe P2 concentration was 0.1 μM, all of which had lower CT values.

[0067] Table 1 Results of fluorescence PCR detection of plasmid pUC19-p72Ⅱ

[0068]

[0069]

[0070] On this basis, the pUC19-p72 I plasmid was used as a template, the p72 F / p72 R primer concentrations were 0.3 μM, 0.4 μM, and 0.5 μM, respectively, and the probe concentration was 0.1-0.5 μM. After combination, the genotype I ASFV p72 gene was detected, and the optimal primer and probe usage amounts were determined. The results are shown in Table 2. The results showed that the best detection effect was achieved when the p72 primer concentration was 0.4 μM each and the probe P1 concentration was 0.1 μM.

[0071] Table 2 Results of fluorescence PCR detection of plasmid pUC19-p72 I

[0072]

[0073] On the basis of the above, primers and probes for detecting the p30Ⅱ gene were added. The qPCR detection results at different primer and probe concentrations are shown in Table 3. The results showed that qPCR had the best detection effect when the concentrations of primers CP204L F / CP204L R were 0.4 μM each and the P3 probe concentration was 0.4 μM.

[0074] Table 3 Results of fluorescence PCR detection of plasmid pBM18A-p30Ⅱ

[0075]

[0076] It can be seen that in the multiplex qPCR reaction system, the detection effect is best when the primers p72 F / p72R are 0.4 μM each, P1 is 0.1 μM, the probe P2 concentration is 0.1 μM, and the primers CP204LF / CP204LR are 0.4 μM each, P3 is 0.4 μM. The specific reaction system is shown in Table 4.

[0077] Table 4 Multiplex fluorescence PCR amplification reaction system

[0078]

[0079]

[0080] 2. Determination of Annealing Temperature for Multiplex Fluorescence PCR

[0081] In a 25 μL multiplex fluorescence PCR reaction system, the reaction solution was prepared according to the reaction system in Table 4 to 22.5 μL, and 2.5 μL (10 8 The positive template plasmids pUC19-p72I, pUC19-p72II, and pBM18A-p30II were mixed in equal proportions (1000 copies / μL) and then centrifuged briefly before testing. The results of multiplex fluorescence PCR are shown in Table 5. The results showed that the CT values ​​for the three target genes were relatively small when the annealing and extension temperature was 59°C. Therefore, the reaction procedure was determined to be: 94°C pre-denaturation for 20 seconds; 98°C denaturation for 5 seconds; 59°C annealing and extension for 30 seconds with fluorescence collection, for a total of 45 cycles.

[0082] Table 5 Determination of annealing temperature

[0083] Annealing temperature 56℃ 57℃ 58℃ 59℃ 60℃ 61℃ pUC19-p72Ⅰ - 17.83 17.759 16.526 17.392 16.335 pUC19-p72Ⅱ - 18.719 18.466 17.992 18.194 18.021 pBM18A-p30Ⅱ - 17.862 18.095 17.936 18.383 -

[0084] Example 4 Sensitivity test and drawing of standard curve

[0085] The plasmid was diluted to a concentration of 10 8 -10 1 The detection limit of the multiplex fluorescence PCR for the plasmid pUC19-p72Ⅰ was 10 2 copies / μL, and the detection limit for pUC19-p72Ⅱ was 10 2 copies / μL, and the detection limit for pBM18A-p30Ⅱ was 10 1 copies / μL( Figure 5-Figure 7 ).

[0086] The standard curve was drawn with the logarithm of the copy number of the plasmid standard as the horizontal axis and the CT value as the vertical axis. According to the test results of the plasmid pUC19-p72Ⅰ, the standard curve was y = -3.2431x + 43.442, and the correlation coefficient R 2 =0.9995, the detection results of plasmid pUC19-p72Ⅱ, the obtained standard curve is y=-3.3455x+44.085, the correlation coefficient R 2 =0.9985, the detection results of plasmid pBM18A-p30Ⅱ, the obtained standard curve is y=-3.3048x+42.985, the correlation coefficient R2=0.9968( Figure 8 The above results showed that there was a good correlation between the target gene concentration and the CT value detected by the multiplex qPCR.

[0087] Example 5 Multiplex fluorescence PCR repeatability test and specificity test

[0088] 1. Repeatability test

[0089] Plasmids pUC19-p72Ⅰ, pUC19-p72Ⅱ, and pBM18A-p30Ⅱ from the same batch and different batches were used as templates, with plasmid concentrations of 10 3 copies / μL, 10 5 copies / μL, 10 7 Multiplex fluorescence PCR was performed using 100 copies / μL of qPCR. The intra- and inter-assay mean, standard deviation, and coefficient of variation (CV) were calculated based on the CT values. As shown in Tables 6 and 7, the intra-assay CV of this qPCR assay ranged from 0.261% to 2.921%, and the inter-assay CV ranged from 0.976% to 3.961%, both less than 5%, demonstrating good reproducibility of the established multiplex fluorescence PCR assay.

[0090] Table 6 Intra-batch repeatability test

[0091]

[0092] Table 7 Inter-batch repeatability test

[0093]

[0094]

[0095] 2. Specificity test

[0096] The established multiplex fluorescence PCR was used to detect known PCV-2, PRRSV, PPV, FMDV, PRV, CSFV, and PEDV nucleic acids. Positive controls of ASFVYNCX-2021-060, ASFV JS, and ASFVNJS23-1 nucleic acids and negative controls of healthy pig tissue DNA were also set up to confirm the detection specificity of the multiplex fluorescence PCR. Figure 9 As shown, only the positive nucleic acid showed a specific amplification curve, while no specific amplification curve appeared in other groups, indicating that the multiplex fluorescent PCR had good detection specificity.

[0097] Example 6 Kit Assembly and Field Sample Detection Test

[0098] Based on the above examples, the components were assembled into a kit, wherein each kit (50 reactions) contained 2× reaction solution (including 125 μL of 10× PCR Buffer, 100 μL of 2.5 mM dNTP Mixture, 50 μL of 0.4 μM primers p72F, p72 R, p30 F, and p30 R, 12.5 μL of 0.1 μM probes P1 and P2, 50 μL of 0.1 μM probe P3, and 125 μL of sterile deionized water without nuclease), 625 μL of TakaraTaq HS (5 U / μL), 12.5 μL of positive control (10 7 126 μL of plasmids pUC19-p72Ⅰ, pUC19-p72, pBM18A-p30Ⅱ (42 μL each) and negative control (10 7 125 μL of plasmid pUC19 (100 copies / μL) and 500 μL of sterile deionized water free of nuclease.

[0099] The reaction system for each reaction was: 12.5 μL of 2× reaction solution, 9.75 μL of sterile deionized water without nuclease, 2.5 μL of template, and 0.25 μL of Takara Taq HS.

[0100] The reaction program was as follows: pre-denaturation at 94°C for 20 s; denaturation at 98°C for 5 s, annealing and extension at 59°C for 30 s and fluorescence collection, for a total of 45 cycles.

[0101] Result determination: When using this multiplex fluorescence PCR kit for detection, if only the FAM channel is detected as positive, it is genotype I ASFV; if both the VIC / ROX channels are detected as positive, it is genotype II ASFV; if both the FAM / ROX channels are detected as positive, it is genotype I / II recombinant ASFV.

[0102] Multiplex fluorescence PCR was performed on 144 samples. As shown in Table 8, the three known genotype I / II recombinant ASFV nucleic acid samples all tested positive for recombinant ASFV I / II; the nucleic acid sample ASFVYNCX-2021-060 tested positive for genotype I ASFV; the nine known genotype II ASFV nucleic acid samples all tested positive for genotype II ASFV; and all 132 ASFV-negative clinical samples tested negative. This indicates that the established multiplex fluorescence PCR assay has a high detection accuracy for samples and can be used for differential diagnosis of different ASFV genotypes in the field.

[0103] Table 8 Field sample multiplex fluorescence PCR kit test results

[0104]

[0105]

[0106]

[0107]

[0108] Although the present application has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications or improvements may be made to the present application. Therefore, such modifications or improvements, without departing from the spirit of the present application, are within the scope of protection claimed in the present application.

Claims

1. A probe for identifying genotype I, genotype II, and genotype I / II recombinant African swine fever virus, characterized in that: It includes SNP typing probes P1 and P2 for genotype I and genotype II ASFV B646L genes, and a specific probe P3 for genotype II ASFV CP204L gene. The nucleotide sequences of P1, P2 and P3 are shown in SEQ ID NO.5-7.

2. The probe according to claim 1, characterized in that The 5' end of P1 is labeled with FAM, the 3' end is labeled with MGB, and the oligonucleotide G at position 7 is a locked nucleotide.

3. The probe according to claim 1, wherein The 5' end of P2 is labeled with VIC, the 3' end is labeled with MGB, and the oligonucleotide A at position 7 is a locked nucleotide.

4. The probe according to claim 1, wherein The 5' end of P3 is labeled with ROX, and the 3' end is labeled with MGB.

5. Primers for identifying genotype I, genotype II, and genotype I / II recombinant African swine fever viruses, characterized in that: The nucleotide sequence of the primer for detecting the ASFV B646L gene in the probe according to claim 1 is shown in SEQ ID NO.1-2.

6. The primer according to claim 5, characterized in that The nucleotide sequence of the primer for detecting the genotype II ASFV CP204L gene is shown in SEQ ID NO.3-4.

7. Use of the probe according to any one of claims 1 to 4 or the primer according to claim 5 or 6 in the preparation of a kit for detecting African swine fever virus.

8. A kit for identifying genotype I, genotype II, and genotype I / II recombinant African swine fever virus, characterized in that: The method comprises a reaction solution, a DNA polymerase, a positive control, a negative control and sterile deionized water without nuclease, wherein the reaction solution comprises the probe according to any one of claims 1 to 4 or the primer according to claim 5 or 6.

9. A method for identifying genotype I, genotype II, and genotype I / II recombinant African swine fever virus, characterized in that: The kit according to claim 8 is used for PCR detection, and the type of African swine fever virus strain is determined according to the fluorescence signal results. If only the FAM channel is detected as positive, it is genotype I ASFV; if both the VIC and ROX channels are detected as positive, it is genotype II ASFV; if both the FAM and ROX channels are detected as positive, it is genotype I / II recombinant ASFV.

10. The method according to claim 9, characterized in that The reaction system for each reaction was: 2×12.5 μL of the reaction solution, 9.75 μL of the nuclease-free sterile deionized water, 2.5 μL of the template, and 0.25 μL of the DNA polymerase. The reaction procedure was: pre-denaturation at 94°C for 20 s; denaturation at 98°C for 5 s, annealing and extension at 59°C for 30 s, and fluorescence collection, for a total of 45 cycles.

Images