A quadruple fluorescent quantitative PCR detection kit for detecting African swine fever

Abstract

The application relates to a quadruple fluorescent quantitative PCR detection kit for detecting African swine fever virus (ASFV) type I, type II wild strains and / or ASFV-G-DI177L strains, which comprises a set of specific primers and fluorescent probes for detecting the African swine fever virus type I, type II wild strains and / or ASFV-G-DI177L strains, the nucleotide sequence of the specific primers is shown in SEQ ID NO. 1, 2, 4, 5, 7, 8, 10 and 11; and the nucleotide sequence of the specific fluorescent probes is shown in SEQ ID NO. 3, 6, 9 and 12. The application designs a quadruple fluorescent quantitative PCR detection method, four genes can be detected at the same time, and the four pairs of primers and probes have relatively consistent amplification efficiency. The application establishes a method for identifying the African swine fever virus gene type I wild strain, the gene type II wild strain and the ASFV-G-DI177L strain based on the characteristics of domestic African swine fever epidemic strains.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, and specifically relates to a quadruple real-time PCR detection kit for detecting African swine fever type I, type II wild-type strains and / or ASFV-G-ΔI177L strain. Background Technology

[0002] African swine fever (ASF) is a highly contagious, acute, and deadly infectious disease caused by the African swine fever virus (ASFV) infecting domestic and wild pigs. Clinically, it is characterized by high morbidity and high mortality. ASFV belongs to the genus *African swine fevervirus* of the family *African swine feverviridae*. It is an icosahedral, enveloped, double-stranded DNA virus. Due to deletions or insertions in the genome sequence, different strains of ASFV have genome sizes ranging from 170 to 190 kb, encoding more than 200 proteins. ASFV has a complex protein and genome structure, is extremely stable in the environment, and its long-term transmission among domestic and wild pigs has led to its genetic diversity. Based on the variable nucleotide sequence at the 3' end of the B646L gene encoding the capsid protein p72, at least 24 ASFV genotypes exist worldwide.

[0003] Through genetic evolutionary analysis, researchers discovered that the African swine fever strain first reported in my country belongs to genotype II. Researchers have summarized and innovated the theoretical and practical experience of ASF and other major animal disease prevention and control. Through precise eradication technology, ASFV-positive pigs can be removed in a timely manner, the spread of the virus can be prevented, and the goal of purifying ASFV in the pig herd can be achieved.

[0004] Published research results indicate that ASFV-G-ΔI177L vaccine administration may result in viral shedding, potentially posing a risk of horizontal transmission. Furthermore, viral titers do not decrease with increasing passage numbers, and offspring become increasingly stable. Given the long production cycle of sows and the fact that animal research experiments only last 28 days, the efficacy and safety of the ASFV-G-ΔI177L vaccine require further investigation.

[0005] The control measures and targeted eradication methods for different strains of ASF vary greatly from farm to farm. Genotype I attenuated strains are difficult to detect early and accurately eliminate. Early detection, early diagnosis, and strain typing are crucial for ASF control. TaqMan real-time quantitative PCR technology has been widely used in the clinical diagnosis of ASF due to its high specificity, high sensitivity, and short processing time. Currently, genotype II virulent strains and genotype I attenuated strains have appeared in my country, and there is a possibility that these strains may enter the I177L gene-deleted vaccine. In production practice, single or mixed infections of different strains may occur. Therefore, it is necessary to establish differential diagnostic methods for these different strain types.

[0006] Current ASFV detection technologies are primarily based on virulent genotype II strains and related partially deleted strains, and cannot differentiate between attenuated genotype I strains and I177L gene-deleted vaccine strains. Therefore, there is an urgent need to establish a rapid, accurate, and simultaneous real-time quantitative PCR method for detecting and identifying multiple African swine fever virus strains. Summary of the Invention

[0007] To address the aforementioned technical problems, the present invention aims to provide a quadruple quantitative fluorescent probe primer combination, detection method, and kit for identifying African swine fever genotype I wild-type strains, genotype II wild-type strains, and ASFV-G-ΔI177L strains.

[0008] This invention provides a set of specific primers and fluorescent probes for detecting African swine fever type I wild-type strain, type II wild-type strain and ASFV-G-ΔI177L strain. The nucleotide sequences of the specific primers are shown in SEQ ID NO. 1, 2, 4, 5, 7, 8, 10 and 11; the nucleotide sequences of the specific fluorescent probes are shown in SEQ ID NO. 3, 6, 9 and 12.

[0009] Furthermore, the 5' end of the probe is modified with different fluorescent groups, and the 3' end is modified with suitable quenching groups.

[0010] Furthermore, the fluorescent group is FAM, VIC, Cy5 and TARMA, and the quenching group is BHQ1, BHQ2, BHQ3 and BHQ2.

[0011] This invention also provides the application of the primer and probe combination in the preparation of a kit for detecting African swine fever type I, type II wild-type strains and ASFV-G-ΔI177L strain.

[0012] The present invention also provides a quadruple real-time PCR detection kit for detecting African swine fever type I, type II wild-type strains and ASFV-G-ΔI177L strain, the kit comprising the primer and probe combination.

[0013] Furthermore, the kit also includes one or more of the following: probe-based real-time PCR premix, positive control, and negative control.

[0014] Furthermore, the positive control contains the B646L gene, I9R gene, F778R gene and ΔI177L inserted gene, and the negative control is ddH2O.

[0015] Furthermore, the PCR amplification reaction system of the detection kit is as follows: 25 μL of probe-based real-time PCR premix, 1 μL each of the primers described in claim 1 (10 μM), 1 μL each of the probes described in claim 1 (10 μM), 4 μL of nucleic acid template, and ddH2O to a final volume of 50 μL.

[0016] Furthermore, the PCR amplification reaction conditions of the detection kit are: 25℃ for 10 min; 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, for 40 cycles.

[0017] The advantages of this invention over the prior art are as follows:

[0018] This invention designs a quadruple real-time PCR detection method that can simultaneously detect four genes, and the four pairs of primers and probes have relatively consistent amplification efficiencies. Based on the characteristics of African swine fever virus strains circulating in China, this invention establishes a method for identifying African swine fever virus genotype I strains, genotype II strains, and ASFV-G-ΔI177L strain.

[0019] This invention designs suitable primers and probes in conserved gene sequences, and ensures that the amplification efficiency of four pairs of primers and probes remains relatively consistent by optimizing primer concentration, probe concentration, and primer-probe ratio.

[0020] Currently, there are type I and type II strains of the virus in China, and the gene sequences of the two strains, I9R and F778R, differ. Primers and probes targeting the I9R and F778R genes were designed to detect the I9R and F778R genes and identify whether the infection is caused by a type I strain, a type II strain, or a mixture of both strains.

[0021] Vietnam has approved the ASFV-G-ΔI177L vaccine for market use. When constructing the ASFV-G-ΔI177L strain, 112 bases of the I177L gene were deleted, and a new sequence was inserted at the deletion site. Specific primers and probes were designed based on the inserted sequence to identify whether it is the ASFV-G-ΔI177L strain.

[0022] This invention can simultaneously identify and detect African swine fever genotype I strains, genotype II strains, and ASFV-G-ΔI177L strains, as well as mixed infections of different strains.

[0023] In this invention, primers and probes for different genes do not interfere with each other, resulting in high detection sensitivity.

[0024] This invention has high specificity and does not undergo specific amplification with common swine disease virus strains.

[0025] This invention provides rapid, efficient, and highly repeatable and stable detection capabilities, and can be used for batch testing. Attached Figure Description

[0026] Figure 1 shows the gene sequence alignment of genotype I and genotype II strains; where A is the alignment of the I9R gene amplification sequence; B is the alignment of the F778R gene amplification sequence; GI: genotype I; GI: genotype II; red bases: bases with differences.

[0027] Figure 2 shows the sequence diagrams of gene plasmid synthesis; where: A is the sequence diagram of B646L gene plasmid synthesis; B is the sequence diagram of I9R gene plasmid synthesis; C is the sequence diagram of F778R gene plasmid synthesis; and D is the sequence diagram of ΔI177L insertion sequence plasmid synthesis.

[0028] Figure 3 The diagram shows the specificity amplification curves for quadruple real-time quantitative PCR. Specifically: 1a–1d shows the amplification curves for the FAM, VIC, Cy5, and TARMA channels of the positive plasmid; 2a–2d shows the amplification curves for the FAM, VIC, Cy5, and TARMA channels of the type I wild-type virus strain; 3a–3d shows the amplification curves for the FAM, VIC, Cy5, and TARMA channels of the type II gene deletion strain; and 4–11 show the amplification curves for PRRSV, CSFV, PRV, PCV2, PCV3, PEDV, TGEV, and the negative control, respectively.

[0029] Figure 4 shows the sensitivity detection results of quadruple real-time quantitative PCR; where: A is the amplification curve of the B646L gene; B is the amplification curve of the I9R gene; C is the amplification curve of the F778R gene; D is the amplification curve of the ΔI177L insert sequence; a~h are 10 7 ~10 0 copies / μL

[0030] Figure 5 shows the results of constructing the quadruple real-time quantitative PCR standard curves; where: A is the standard curve of the B646L gene; B is the standard curve of the I9R gene; C is the standard curve of the F778L gene; and D is the standard curve of the ΔI177L insertion sequence.

[0031] Figure 6 shows the amplification curves of clinical samples detected by the quadruple real-time quantitative PCR method; where: A is the genotype I wild-type strain; B is the genotype II wild-type strain; 1a-1d are the amplification curves of the positive control; 2a-2d are the amplification curves of the clinical samples; and 3a-3d are the amplification curves of the negative control. Detailed Implementation

[0032] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0033] Example 1

[0034] 1. Design of specific primers and probes

[0035] Currently, the main circulating strains of ASFV in China are genotype I and II, and the I9R and F778R gene sequences of the two strains differ. Specific primers and probes were designed by comparing the I9R and F778R genes of the prevalent genotype I and II strains both domestically and internationally, focusing on the differing but conserved regions. The alignment results of the designed primer amplification sequences with the two genotypes are shown in Figure 1. Specific primers and probes were designed based on the sequence inserted during the design of ASFV-G-ΔI177L. Specific sequences are shown in Table 1.

[0036] Genotype I and Genotype II strains differ in the I9R and F778R gene sequences. Specific primers and probes designed in the differing and conserved regions can differentiate between Genotype I and Genotype II strains. Specific primers and probes designed based on the sequences inserted during the construction of the ASFV-G-ΔI177L strain can be used to identify whether it is the ASFV-G-ΔI177L strain.

[0037] Table 1 Primer and probe sequences

[0038]

[0039] 2. Preparation of plasmid standards

[0040] Based on the sequences of Chinese ASFV isolates (MK333180 and MZ945536) and ASFV-G-ΔI177L strain in GenBank, gene sequences B646L, I9R, F778R, and ΔI177L were synthesized (see Figure 2 for specific sequences). These sequences were cloned into the pUC57 vector, transformed into DH5α competent bacterial cells, and plasmids were extracted. After sequencing verification, they were used as plasmid standards for multiplex quantitative PCR.

[0041] 3. Quantitative Real-Time PCR Detection Method

[0042] (1) PCR amplification reaction

[0043] Nucleic acid extracted from samples such as pharyngeal swabs was used as a template, plasmid standard was used as a positive control, and ddH2O was used as a negative control. The amplification reaction system was as follows: 25 μL of PCR premix, 1 μL of each primer, 1 μL of each probe, 4 μL of nucleic acid template, and ddH2O to a final volume of 50 μL. The amplification reaction conditions were as follows: 25℃ for 10 min; 95℃ for 30 s; 95℃ for 5 s; 60℃ for 30 s, for 40 cycles. Fluorescence signals were collected during amplification at 60℃.

[0044] (2) Result determination

[0045] For a positive control, all four channels must have Ct values ​​<35 and exhibit specific amplification curves. For a negative control, no Ct value and no specific amplification curve are required. Both conditions must be met for the experimental result to be valid. The presence of a FAM signal is necessary to confirm a positive result for African swine fever virus nucleic acid. If no FAM signal is present but other signals are present, the experiment is invalid and retesting is required.

[0046] ① When the FAM and VIC fluorescent signal channels have signals and specific amplification curves, while the Cy5 and TARMA fluorescent signal channels have no signals or no amplification curves, it is determined to be a type I wild-type strain.

[0047] ② When the FAM and Cy5 fluorescent signal channels have signals and specific amplification curves, while the VIC and TARMA fluorescent signal channels have no signals or no amplification curves, it is determined to be a type II wild-type strain.

[0048] ③ When the FAM and TARMA fluorescent signal channels have signal and specific amplification curves, and the VIC and Cy5 fluorescent signal channels have no signal and specific amplification curves, it is determined to be the ASFV-G-ΔI177L strain.

[0049] ④ When the FAM, VIC, and Cy5 fluorescent signal channels show signal and specific amplification curves, and the TARMA non-fluorescent signal channel shows signal and specific amplification curves, it is determined to be a mixed infection of type I wild-type virus strain and type II wild-type virus strain.

[0050] ⑤ When the FAM, VIC, and TARMA fluorescent signal channels show signal and specific amplification curves, and the Cy5 non-fluorescent signal channel shows signal and specific amplification curves, it is determined to be a mixed infection of type I wild-type virus strain and ASFV-G-ΔI177L virus strain.

[0051] ⑥ When the FAM, Cy5, and TARMA fluorescent signal channels show signal and specific amplification curves, and the VIC non-fluorescent signal channel shows signal and specific amplification curves, it is determined to be a mixed infection of type II wild-type virus strain and ASFV-G-ΔI177L virus strain.

[0052] ⑦ When the FAM, VIC, Cy5 and TARMA fluorescent signal channels all show signal and specific amplification curves, it is determined to be a mixed infection of type I wild-type virus strain, type II wild-type virus strain and ASFV-G-ΔI177L strain.

[0053] 4. Specificity test

[0054] Using genotype I and genotype II wild-type viruses, as well as DNA or cDNA of common pig farm viruses such as porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis virus (TGEV) as templates, quantitative real-time PCR was performed using the above-mentioned reaction system and procedure.

[0055] The results are as follows Figure 3 As shown, the detection results for PRRSV, CSFV, PRV, PCV2, PCV3, PEDV, and TGEV, as well as the negative control, were all negative, indicating that the method has good specificity and no cross-reaction with common swine viral disease pathogens, and can be applied to clinical detection.

[0056] 5. Sensitivity test

[0057] The standard plasmid was serially diluted 10-fold to a concentration of 10. 7 copies / μL~10 0 The plasmid was diluted to copies / μL and used in the above reaction system and procedure. The diluted plasmid was used as a template for real-time PCR detection, and the standard curve was analyzed at the same time.

[0058] The results are shown in Figure 4. The minimum copy number of positive standard plasmids that could be detected by quadruple real-time PCR was 1 copy, 1 copy, 1 copy, and 1 copy, indicating that the method has high sensitivity. The results are shown in Figure 5. Within the dilution range, the template amount and Ct value showed a good linear relationship, with a correlation coefficient R0. 2 All are greater than 0.99.

[0059] 6. Repeatability test

[0060] The standard plasmid was serially diluted 10-fold to a concentration of 10. 7 ~10 2 Using the above reaction system and procedure, the diluted plasmid was used as a template for real-time PCR detection, with three replicates for each gradient.

[0061] The results show that the repeatability of each gradient is very good, and the coefficient of variation (CV value) is less than 2%, indicating that the method has good repeatability.

[0062] 7. Clinical sample testing

[0063] Using clinically collected DNA samples as templates, positive plasmids as positive controls, and ddH2O as negative controls, parallel B646L gene detection was performed using the method established in this study and the method recommended by WOAH. The consistency of ASFV detection positive results between the two methods was compared. Following the ASFV genotyping method recommended by WOAH, the B646L gene in ASFV-positive samples was amplified using conventional PCR, then sent to a company for sequencing. Homology analysis was performed on the obtained sequences to further clarify the strain type of each positive sample, and the consistency of genotyping results between the two methods was compared.

[0064] According to the judgment criteria, the results are shown in Table 2 and Figure 6. A total of 10 positive samples were detected using this method, including 2 type I wild-type strains and 8 type II wild-type strains. ASFV-G-ΔI177L was not detected. The positive samples and strain types detected in this study are consistent with the detection and typing methods recommended by WOAH, indicating that this method can achieve the goal of sensitive, rapid, and accurate detection of different African swine fever virus strains.

[0065] Table 2 Clinical Sample Testing Results

[0066] strain type This method WOAH Recommended Method ASFV negative 1216 1216 ASFV positive 10 10 Type I wild-type strain 2 2 Type II wild-type strain 8 8 ASFV-G-ΔI177L 0 0

Claims

1. A set of specific primers and fluorescent probes for simultaneous detection of African swine fever type I and II wild-type strains and / or ASFV-G-ΔI177L strain, characterized in that, The nucleotide sequences of the specific primers are shown in SEQ ID NO. 1, 2, 4, 5, 7, 8, 10, and 11; the nucleotide sequences of the specific fluorescent probes are shown in SEQ ID NO. 3, 6, 9, and 12.

2. The specific primer and fluorescent probe combination according to claim 1, characterized in that, The probe is modified with different fluorescent groups at its 5' end and with corresponding quenching groups at its 3' end.

3. The specific primer and fluorescent probe combination according to claim 2, characterized in that, The fluorescent groups are FAM, VIC, Cy5 and TARMA, and the quenching groups are BHQ1, BHQ2, BHQ3 and BHQ2.

4. The use of the specific primer and fluorescent probe combination described in claim 1 in the preparation of a kit for detecting African swine fever type I, type II wild-type strains and / or ASFV-G-ΔI177L strain.

5. A quadruple real-time PCR detection kit for simultaneously detecting African swine fever type I and II wild-type strains and / or ASFV-G-ΔI177L strain, characterized in that, The kit comprises the primer and probe combination as described in claim 1.

6. The reagent kit according to claim 5, characterized in that, The kit also includes one or more of the following: probe-based quantitative PCR premix, positive control, and negative control.

7. The reagent kit according to claim 6, characterized in that, The positive control contains the genes B646L, I9R, F778R, and I177L, and the negative control is ddH2O.

8. The kit according to any one of claims 5 to 7, characterized in that, The PCR amplification reaction system of the detection kit is as follows: 25 μL of probe-based real-time PCR premix, 1 μL each of the primers described in claim 1 (10 μM), 1 μL each of the probes described in claim 1 (10 μM), 4 μL of nucleic acid template, and ddH2O to a final volume of 50 μL.

9. The kit according to any one of claims 5 to 7, characterized in that, The PCR amplification reaction conditions for the detection kit are: 25℃ for 10 min; 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, for 40 cycles.

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