Primer probe combination, method and application for identifying genotype of african swine fever virus

Abstract

The application provides a primer probe combination for identifying the genotype of African swine fever virus, comprising a primer probe combination a for identifying a B646L gene, a primer probe combination b for identifying an MGF-110-5L gene and a primer probe combination c for identifying an I177L gene. The method for identifying the genotype of African swine fever virus by using the primer probe combination is as follows: extracting the genomic DNA of a sample to be detected, using the extracted genomic DNA of the sample to be detected as a template, performing a triple fluorescent quantitative PCR reaction by using the primer probe combination, and judging the genotype of African swine fever virus according to the fluorescent signal. The application also provides the application of the primer probe combination to the preparation of a kit for identifying the genotype of African swine fever virus. The concentration and reaction conditions of the primer probe combination are optimized, so that the designed primer probe combination has strong specificity, sensitivity and repeatability, not only solves the identification problem of African swine fever virus, but also simultaneously realizes the synchronous identification of three kinds of epidemic strain genotypes.

Description

Technical Field

[0001] This invention belongs to the field of African swine fever virus technology, specifically relating to a primer-probe combination, method, and application for identifying African swine fever virus genotypes. Background Technology

[0002] This study utilizes the differences in the B646L, MGF-110-5L, and I177L genes of African swine fever virus (ASFV) types I, II, and gene deletion types to identify the genotype of ASFV. Currently, there is no quantitative real-time PCR method for identifying ASFV genotypes using the MGF-110-5L gene. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a primer-probe combination, method and application for identifying African swine fever virus genotypes, which addresses the shortcomings of the prior art. After condition optimization, the primer-probe combination has strong specificity, sensitivity and repeatability, and no cross-reaction with other viruses. It not only solves the problem of African swine fever virus identification, but also enables the simultaneous identification of genotypes of three circulating strains.

[0004] To solve the above technical problems, the technical solution adopted by the present invention is: a primer and probe combination for identifying the genotype of African swine fever virus, wherein the primer and probe combination includes primer and probe combination a for identifying the B646L gene, primer and probe combination b for identifying the MGF-110-5L gene, and primer and probe combination c for identifying the I177L gene. The primer-probe combination a includes an upstream primer a, a downstream primer a, and a probe a. The nucleotide sequences of the upstream primer a and the downstream primer a are shown in SEQ ID No: 1-2, respectively. The nucleotide sequence of the probe a is as follows: 5'-FAM-TTGCGAGGAAACGTTTGAAGCTGC-BHQ1-3'; The primer-probe combination b includes an upstream primer b, a downstream primer b, and a probe b. The nucleotide sequences of the upstream primer b and the downstream primer b are shown in SEQ ID No: 3-4, respectively. The nucleotide sequence of the probe b is as follows: 5'-VIC-TTGTGAGGTACTGGATCAGAACAATCC-BHQ1-3'; The primer-probe combination c includes an upstream primer c, a downstream primer c, and a probe c. The nucleotide sequences of the upstream primer c and the downstream primer c are shown in SEQ ID No: 5-6, respectively. The nucleotide sequence of the probe c is as follows: 5'-CY5-TTGTGAGGTACTGGATCAGAACAATCC-BHQ3-3'.

[0005] The method for identifying African swine fever virus genotype using primer-probe combinations of the present invention is as follows: S1. Extract genomic DNA from the sample to be tested; S2. Using the genomic DNA of the sample to be tested extracted in S1 as a template, a triple fluorescence quantitative PCR reaction is performed using the primer and probe combination described above. S3. Determine the African swine fever virus genotype based on fluorescence signals.

[0006] Preferably, the method for determining the genotype of African swine fever virus in S3 is as follows: when the amplification curves of the B646L gene, I177L gene, and MGF-110-5L gene are detected, the sample to be tested contains African swine fever virus type I; when only the amplification curves of the B646L gene and I177L gene are detected, the sample to be tested contains African swine fever virus type II.

[0007] The present invention also provides the application of the above-mentioned primer-probe combination for identifying African swine fever virus genotypes, wherein the primer-probe combination for identifying African swine fever virus genotypes is used to prepare a kit for identifying African swine fever virus genotypes.

[0008] Compared with the prior art, the present invention has the following advantages: 1. To distinguish between three ASFV genotypes—genotype I, type II, and the I177L gene deletion strain—this invention utilizes triple real-time PCR. Based on the B646L, MGF-110-5L, and I177L genes of ASFV virus strains reported in the GenBank database, specific probes and primers were designed. After optimization of reaction conditions and verification of specificity, sensitivity, and repeatability, the results show that this method exhibits good specificity, sensitivity, and repeatability, and shows no cross-reactivity with other viruses. Furthermore, the detection limit for MGF-110-5L and I177L positive plasmids is [not specified in the original text]. The detection limit for the B646L positive plasmid is [missing information]. The coefficients of variation between and within groups were both <1%. In summary, the establishment of this triplet real-time PCR method not only solved the problem of African swine fever virus identification, but also enabled the simultaneous identification of the genotypes of three circulating strains.

[0009] 2. This invention is the first to screen MGF-110-5L as a gene to distinguish between African swine fever type I and type II, providing a new method for more accurate identification and typing of African swine fever virus by real-time quantitative PCR.

[0010] 3. This invention can not only effectively distinguish the I177L gene deletion strain, but also, for the first time, identify and distinguish the type I and type II strains by using the target gene MGF110-5L. The MGF110-5L specific primer probe can also be used as a DIVA (distinguishing between infection and vaccination) marker for gene deletion attenuated live vaccine candidate strains under investigation (such as ASFV-G-ΔMGF110-5L-6L).

[0011] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. Attached Figure Description

[0012] Figure 1 This is an optimized amplification curve of the B646L gene primer concentration in Example 2 of the present invention.

[0013] Figure 2 This is an optimized amplification curve of the I177L gene primer concentration in Example 2 of the present invention.

[0014] Figure 3 This is an amplification curve of the MGF gene with optimized primer concentration in Example 2 of the present invention.

[0015] Figure 4 This is the concentration-optimized amplification curve of probe a B646L in Example 2 of the present invention.

[0016] Figure 5 This is an optimized amplification curve of MGF probe b concentration in Example 2 of the present invention.

[0017] Figure 6 This is the concentration-optimized amplification curve of the I177L probe c in Example 2 of the present invention.

[0018] Figure 7 This is the amplification curve of the annealing temperature of 52.5℃ in Example 2 of the present invention.

[0019] Figure 8 This is an amplification curve of annealing temperature 55°C in Example 2 of the present invention.

[0020] Figure 9 This is the amplification curve of the annealing temperature of 57.5℃ in Example 2 of the present invention.

[0021] Figure 10 This is an amplification curve of annealing temperature 60°C in Example 2 of the present invention.

[0022] Figure 11 This is a specificity verification amplification curve from Example 3 of the present invention.

[0023] Figure 12 This is the standard curve diagram of the B646L gene in Example 3 of the present invention.

[0024] Figure 13 This is the standard curve diagram of the I177L gene in Example 3 of the present invention.

[0025] Figure 14 This is the standard curve diagram of the MGF gene in Example 3 of the present invention.

[0026] Figure 15 This is the sensitivity verification amplification curve in Example 3 of the present invention.

[0027] Figure 16 This is a repeatability verification amplification curve diagram from Example 3 of the present invention.

[0028] Figure 17 This is the amplification curve of the clinical sample validation in Example 4 of the present invention. Detailed Implementation

[0029] Example 1

[0030] This embodiment describes the design of primer-probe combinations for identifying the genotype of African swine fever virus.

[0031] Based on sequence alignment of the ASFV (African Swine Fever Virus) genotype I and genotype II genome sequences published in NCBI GenBank, and utilizing the differences in the MGF-110-5L gene, primer-probe combination a for identifying the B646L gene, primer-probe combination b for identifying the MGF-110-5L gene, and primer-probe combination c for identifying the I177L gene were designed using Primer Premier 5.0 software. Primer-probe combination a includes an upstream primer a, a downstream primer a, and probe a. The nucleotide sequences of the upstream primer a and the downstream primer a are shown in SEQ ID No: 1-2, respectively. The nucleotide sequence of probe a is: 5'-FAM-TTGCGAGGAAACGTTTGAAGCTGC-BHQ1-3'. Primer-probe combination b includes an upstream primer b, a downstream primer b, and probe b. The nucleotide sequences of the upstream primer b and the downstream primer b are shown in SEQ ID No: 1-2, respectively. As shown in SEQ ID Nos. 3-4, the nucleotide sequence of probe b is: 5'-VIC-TTGTGAGGTACTGGATCAGAACAATCC-BHQ1-3'; the primer-probe combination c includes upstream primer c, downstream primer c, and probe c, the nucleotide sequences of upstream primer c and downstream primer c are shown in SEQ ID Nos. 5-6, and the nucleotide sequence of probe c is: 5'-CY5-TTGTGAGGTACTGGATCAGAACAATCC-BHQ3-3'; after online BLAST alignment, the optimal combination with strong fluorescence increase, high sensitivity, and no interference between each other was screened through combination experiments and synthesized by Sangon Biotech Co., Ltd. Example 2

[0032] This embodiment focuses on optimizing the detection conditions.

[0033] 1. Optimization of primer and probe concentrations: Using the controlled variable method, primers were set with a gradient of 125 nM, and five gradients were set (250 nM, 375 nM, 500 nM, 625 nM, 750 nM). Plasmids (pUC57-B646L, pUC57-MGF-110-5L, and pUC57-I177L, synthesized and sequenced by Sangon Biotech (Shanghai) Co., Ltd.) were used as templates (medium-strong positive). The plasmid dilution was 10-1. -5 qPCR experiments were performed to determine the optimal primer concentration. Using the optimal primer concentration, probes were set at a gradient of 100 nM, with three gradients (150 nM, 250 nM, 350 nM). Using the plasmid as a template (medium-strong positive), qPCR experiments were performed to determine the optimal probe concentration.

[0034] The results of primer concentration optimization are shown in Tables 1-3, and the amplification curves are shown in Tables 1-3. Figures 1-3 As shown, the optimal primer concentration for the B646L gene is 625 nM, the optimal primer concentration for the I177L gene is 500 nM, and the optimal primer concentration for the MGF-110-5L (MGF) gene is 375 nM. At these concentrations, the primer amplification curves exhibit good overall linearity, the lowest CT value, and moderate fluorescence increment.

[0035] Table 1. Primer concentration optimization for the ASFV B646L gene. Table 2. Primer concentration optimization for the ASFV I177L gene. Table 3. Primer concentration optimization for the ASFV MGF-110-5L gene. The results of probe concentration optimization are shown in Tables 4-6, and the amplification curves are shown in Figures 4-6. Figures 4-6 As shown, the optimal concentration of B646L gene probe a is 250 nM, the optimal concentration of MGF-110-5L (MGF) gene probe b is 350 nM, and the optimal concentration of I177L gene probe c is 350 nM. At these concentrations, the probe amplification curves exhibit good overall linearity, the lowest CT value, and moderate fluorescence increment.

[0036] Table 4. Concentration optimization of ASFV B646L probe a Table 5. Concentration optimization of ASFV MGF probe b Table 6. Concentration optimization of ASFV I177L probe c 2. Optimization of annealing temperature: Using the controlled variable method, primers and probes were selected at the optimal concentrations mentioned above. Four temperature gradients were set (52.5℃, 55℃, 57.5℃, 60℃). Using plasmid as a template, two concentrations were set (medium-strong positive and weak positive), with plasmid dilutions of 10⁻⁶ each. -5 and 10 -7 ), and perform qPCR experiments to determine the optimal annealing temperature.

[0037] The results of the annealing temperature optimization are shown in Tables 7-9, and the amplification curves are shown in Tables 7-9. Figures 7-10As shown, there was no significant difference in the overall CT values ​​of the four annealing temperatures. The linearity at 60℃ was less concentrated than that at other annealing temperatures. The medium-strength anode template performed best at 52.5℃, while the weak anode templates performed well at 55℃ and 57.5℃. Therefore, considering the performance at different concentrations, 55℃ was selected as the optimal annealing temperature.

[0038] Table 7 Optimization of Annealing Temperature for ASFV B646L Table 8 Optimization of ASFV MGF Annealing Temperature Table 9 Optimization of annealing temperature for ASFV I177L Example 3

[0039] This embodiment is a verification of the identification effect of the primer-probe combination designed in Example 1 for identifying the genotype of African swine fever virus.

[0040] 1. Specificity: Quantitative real-time PCR was performed using porcine circovirus type 2-WH strain vaccine (PCV2 WH strain vaccine), porcine pseudorabies HB live vaccine, porcine parvovirus WH strain vaccine, foot-and-mouth disease type O vaccine (FMDV-O vaccine), classical classical swine fever live vaccine, porcine parvovirus samples (porcine PPV samples), classical classical swine fever samples (CSFV sample-3), porcine reproductive and respiratory syndrome virus (PRRSV) American strain samples, swine streptococcus samples, Staphylococcus aureus standards, Escherichia coli standards, as well as negative controls (whole blood from healthy pigs) and positive controls (positive plasmid, P triplet), and the detection results were compared.

[0041] The specific steps are as follows: Extract the viral genomic DNA using the Novizan kit; use the extracted genomic DNA as a template; and perform triplet real-time PCR using the primer and probe combination described in Example 1. The PCR reaction system is as follows: DNA template... upstream primer Downstream primers probe upstream primer Downstream primers probe upstream primer Downstream primers probe Premixed solution (Yisheng Biotechnology) ddH2O replenished to The PCR reaction procedure was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 5 s; 55℃ annealing extension for 40 s (fluorescence collection), for a total of 40 cycles.

[0042] The results are as follows Figure 11 As shown, this method does not exhibit nonspecific amplification of porcine circovirus, pseudorabies virus, porcine parvovirus, foot-and-mouth disease virus type O, classical swine fever virus, porcine reproductive and respiratory syndrome virus, streptococcus suis, Staphylococcus aureus, Escherichia coli, or the porcine genome.

[0043] 2. Plotting the standard curve for positive plasmids: The positive plasmid was serially diluted 10-fold to a concentration of [missing value]. , , , , , , , Using the optimized detection method, a standard curve is automatically generated by the real-time PCR instrument.

[0044] The results are as follows Figures 12-14 As shown, the standard curve for B646L is Y = -3.578X + 38.274, R... 2 =0.997, amplification efficiency of 90.31%; I177L standard curve is Y=-3.57X+38.548, R 2 =0.997, amplification efficiency of 90.59%; MGF-110-5L standard curve is Y=-3.585X+38.482, R 2 =0.996, amplification efficiency of 90.09%.

[0045] 3. Sensitivity: The positive plasmid was serially diluted 10-fold to a concentration of [missing value]. , , , , , , , The method was repeated three times for each sample to determine its sensitivity.

[0046] The test results are as follows Figure 15 As shown, after 10-fold serial dilutions, different concentrations of plasmid amplified in a gradient manner, with B646L showing the following results: Amplification was possible at all concentrations, with a positive rate of 100%. I177L and MGF-110-5L were also observed. The positive rate at the specified concentration was 66.67%, meaning one replicate was negative. The limit of detection (LOD) for this method on MGF-110-5L and I177L positive plasmids was [missing value]. The detection limit for the B646L positive plasmid is [missing information]. .

[0047] 4. Repeatability: With concentrations of , The plasmid was used as a template, and the template was repeated 6 times for each dilution. The Ct value for each concentration was calculated, and the results were statistically analyzed to calculate the coefficient of variation (CV).

[0048] The results are shown in Table 10 and Figure 16 As shown, under moderate to weak positive concentrations, the CV values ​​for B646L within groups were 2.000% and 0.284%, and the CV value between groups was 1.973%; for I177L, the CV values ​​were 1.585% and 0.406%, and the CV value between groups was 1.585%; for MGF, the CV values ​​were 1.494% and 0.403%, and the CV value between groups was 1.494%. Under weak positive concentrations, the CV values ​​for B646L within groups were 1.332% and 0.352%, and the CV value between groups was 1.236%; for I177L, the CV values ​​were 0.920% and 0.299%, and the CV value between groups was 1.333%; for MGF, the CV values ​​were 0.607% and 0.297%, and the CV value between groups was 0.959%, ​​all less than 5%; therefore, the method has good reproducibility.

[0049] Table 10 Repeatability Analysis Example 4

[0050] This embodiment uses the primer-probe combination designed in Example 1 to identify the African swine fever virus genotype.

[0051] The nucleic acid of six clinical samples of African swine fever that had been genotyped was detected using multiplex quantitative PCR, including two type I samples and four type II samples. The PCR reaction system and procedure were the same as in Example 3.

[0052] The results are as follows Figure 17 As shown, the positive detection and genotyping concordance rate was 100%. Among them, the two type I samples were positive for the B646L, I177L, and MGF-110-5L genes; the four known type II samples were positive for the B646L and I177L genes, but negative for MGF-110-5L. The clinical sample concordance rate of this method was 100%, indicating that the method established in this study can be used for the detection of clinical samples.

[0053] In summary, the primer-probe combination of this invention has been optimized in terms of primer concentration, probe concentration, and reaction conditions, and its specificity, sensitivity, and repeatability have been verified. The limit of detection (LOD) for MGF-110-5L and I177L positive plasmids is [insert value here]. The detection limit for the B646L positive plasmid is [missing information]. With coefficients of variation of <1% for both inter- and intra-group samples, this primer-probe combination not only solves the problem of African swine fever virus identification but also enables simultaneous identification of the genotypes of three prevalent strains. It can be used to prepare kits for identifying African swine fever virus genotypes. The MGF110-5L specific primer probe can also be used as a DIVA (distinguishing between infection and vaccination) marker for gene-deleted attenuated live vaccine candidate strains under investigation (such as ASFV-G-ΔMGF110-5L-6L).

[0054] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Any simple modifications, alterations, and equivalent changes made to the above embodiments based on the inventive essence shall still fall within the protection scope of the present invention.

Claims

1. A primer-probe combination for identifying the genotype of African swine fever virus, characterized in that, The primer-probe combination includes primer-probe combination a for identifying the B646L gene, primer-probe combination b for identifying the MGF-110-5L gene, and primer-probe combination c for identifying the I177L gene. The primer-probe combination a includes an upstream primer a, a downstream primer a, and a probe a. The nucleotide sequences of the upstream primer a and the downstream primer a are shown in SEQ ID No: 1-2, respectively. The nucleotide sequence of the probe a is as follows: 5'-FAM-TTGCGAGGAAACGTTTGAAGCTGC-BHQ1-3'; The primer-probe combination b includes an upstream primer b, a downstream primer b, and a probe b. The nucleotide sequences of the upstream primer b and the downstream primer b are shown in SEQ ID No: 3-4, respectively. The nucleotide sequence of the probe b is as follows: 5'-VIC-TTGTGAGGTACTGGATCAGAACAATCC-BHQ1-3'; The primer-probe combination c includes an upstream primer c, a downstream primer c, and a probe c. The nucleotide sequences of the upstream primer c and the downstream primer c are shown in SEQ ID No: 5-6, respectively. The nucleotide sequence of the probe c is as follows: 5'-CY5-TTGTGAGGTACTGGATCAGAACAATCC-BHQ3-3'.

2. A method for identifying African swine fever virus genotypes using a primer-probe combination as described in claim 1, characterized in that, The method is as follows: S1. Extract genomic DNA from the sample to be tested; S2. Using the genomic DNA of the sample to be tested extracted in S1 as a template, a triple fluorescence quantitative PCR reaction is performed using the primer and probe combination described above. S3. Determine the African swine fever virus genotype based on fluorescence signals.

3. The method according to claim 2, characterized in that, The method for determining the genotype of African swine fever virus described in S3 is as follows: when the amplification curves of the B646L gene, I177L gene, and MGF-110-5L gene are detected, the sample to be tested contains African swine fever virus type I; when only the amplification curves of the B646L gene and I177L gene are detected, the sample to be tested contains African swine fever virus type II.

4. The application of the primer-probe combination for identifying the genotype of African swine fever virus as described in claim 1, characterized in that, The primer-probe combination for identifying African swine fever virus genotypes is used to prepare a kit for identifying African swine fever virus genotypes.

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