PCR (Polymerase Chain Reaction) primer probe group and method for detecting classical swine fever virus vaccine strain C and classical swine fever virus wild strain

Abstract

The invention discloses a PCR (Polymerase Chain Reaction) primer probe group and a method for detecting a classical swine fever virus vaccine strain C and a classical swine fever virus wild strain, and belongs to the technical field of classical swine fever virus detection. The PCR primer probe group is composed of primers and probes of E2 and NS5A, the primers of E2 and the probes are used for detecting a classical swine fever virus vaccine C strain, and the primers of NS5A and the probes are used for detecting a classical swine fever virus wild strain. The detection method provided by the invention is high in sensitivity, the lowest detection lower limit of an in-vitro transcription pUC57-E2 RNA standard substance is 11.75 copies / [mu] L, and the lowest detection lower limit of an in-vitro transcription pUC57-NAS5A RNA standard substance is 39.81 copies / [mu] L; the specificity is good, and only target viruses are detected; the repeatability is good, and the intra-group variable coefficient and the inter-group variable coefficient are both smaller than 5%.

Description

Technical Field

[0001] The present invention belongs to the technical field of classical swine fever virus detection, and particularly relates to a PCR primer-probe set and method for detecting classical swine fever virus vaccine strain C and classical swine fever virus wild strain. Background Art

[0002] Classical swine fever is a highly contagious disease of pigs caused by classical swine fever virus, which will cause huge economic losses to the pig industry. Existing detection methods cannot distinguish between classical swine fever virus vaccine strain C and wild virus infection, which greatly limits the process of classical swine fever eradication.

[0003] Currently, the commonly used detection methods for classical swine fever mainly include: virus isolation, serological methods and molecular biology detection methods. Nucleic acid detection of classical swine fever virus is the main method for diagnosing classical swine fever in China, mainly including polymerase chain reaction (PCR), real-time fluorescence quantitative PCR technology, and DNA sequencing technology. Among them, the fluorescence quantitative PCR method for differentiating CSFV has the advantages of high sensitivity, fast speed, directly targeting the target virus, and can determine the degree of virus infection through relative abundance. However, for the detection of virus nucleic acid by the fluorescence quantitative PCR method, the samples are mainly serum, swabs, tissue homogenates, etc., and nucleic acid extraction is required for these samples. It generally takes 4-5 hours from sample extraction to confirmation of the detection result, with relatively low efficiency, and false negatives and false positives cannot be evaluated.

[0004] In summary, developing a rapid, accurate and sensitive detection method for differentiating classical swine fever virus vaccine strain C and classical swine fever wild strain is an urgent problem to be solved at present. Summary of the Invention

[0005] Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide a PCR primer-probe set and method for detecting classical swine fever virus vaccine strain C and classical swine fever virus wild strain, so as to solve the problem that the prior art cannot distinguish between classical swine fever virus vaccine strain C and classical swine fever virus wild strain infection.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: A PCR primer-probe set for detecting classical swine fever virus vaccine strain C and classical swine fever virus wild strain is provided, including specific primers and specific probes; the specific primers include specific primers for the CSFV E2 gene of classical swine fever virus vaccine strain C and specific primers for detecting the CSFV NS5A gene of classical swine fever virus wild strain; the specific primers for detecting the CSFV E2 gene include a forward primer CSFV-C-F and a reverse primer CSFV-C-R, the nucleotide sequence of the forward primer CSFV-C-F is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer CSFV-C-R is shown in SEQ ID NO.2; the specific primers for detecting the CSFV NS5A gene include a forward primer CSFV-W-F and a reverse primer CSFV-W-R, the nucleotide sequence of the forward primer CSFV-W-F is shown in SEQ ID NO.4, and the nucleotide sequence of the reverse primer CSFV-W-R is shown in SEQ ID NO.5; the specific probes include a specific probe CSFV-C-P for the CSFV E2 gene of classical swine fever virus vaccine strain C and a specific probe CSFV-W-P for the CSFV NS5A gene of classical swine fever virus wild strain, the nucleotide sequence of CSFV-C-P is shown in SEQ ID NO.3, and the nucleotide sequence of CSFV-W-P is shown in SEQ ID NO.6.

[0007] Based on the above technical solution, the present invention can also be improved as follows: Further, the 5' end of the specific probes CSFV-C-P and CSFV-W-P is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quenching group.

[0008] Further, the fluorescent reporter group labeled at the 5' end of the specific probe CSFV-C-P is FAM, and the fluorescent quenching group labeled at the 3' end is BHQ1.

[0009] Further, the fluorescent reporter group labeled at the 5' end of the specific probe CSFV-W-P is VIC, and the fluorescent quenching group labeled at the 3' end is BHQ1.

[0010] The present invention also provides a kit for detecting classical swine fever virus vaccine strain C and classical swine fever virus wild strain, and the kit includes the above PCR primer-probe set.

[0011] The present invention also provides a method for detecting classical swine fever virus vaccine strain C and classical swine fever virus wild strain, including the following steps: (1) Extract the DNA / RNA of the sample to be tested, and use the DNA / RNA of the sample to be tested as a template; (2) Prepare an amplification reaction solution using the above PCR primer-probe set, and perform a dual fluorescence quantitative RT-PCR reaction; (3) Collect the fluorescence signals of the two channels and make a result determination.

[0012] Based on the above technical solutions, the present invention can also be improved as follows: Further, the above amplification reaction solution includes the following components: template, 2×One Step U+Mix, One Step U+Enzyme Mix, forward primer CSFV-C-F, reverse primer CSFV-C-R, specific probe CSFV-C-P, forward primer CSFV-W-F, reverse primer CSFV-W-R, specific probe CSFV-W-P, and deionized water.

[0013] Further, the concentrations of the forward primer CSFV-C-F, reverse primer CSFV-C-R, specific probe CSFV-C-P, forward primer CSFV-W-F, reverse primer CSFV-W-R, and specific probe CSFV-W-P in the amplification reaction solution are all 400 nmol / L.

[0014] Further, the procedure of the above dual fluorescence quantitative RT-PCR reaction is: 55°C for 15 min, pre-denaturation at 95°C for 2 min, denaturation at 95°C for 5 s, annealing at 60°C for 35 s, 40 cycles.

[0015] Further, the specific determination method of the above step (3) is: when an amplification curve is detected for the E2 gene, it is judged as the classical swine fever virus vaccine strain C; when an amplification curve is detected for the NS5A gene, it is judged as the classical swine fever virus wild strain.

[0016] The present invention has the following beneficial effects: 1. The present invention can simultaneously detect the E2 and NS5A of classical swine fever virus in one reaction, not only can it know whether there is an infection of classical swine fever, but also can distinguish between the classical swine fever virus vaccine strain C and the wild strain, which is time-saving and efficient.

[0017] 2. The amplification of the present invention for the two targets does not interfere with each other, and has strong specificity, high sensitivity, and good repeatability, fully ensuring the amplification of each target and ensuring the high sensitivity of each target.

[0018] 3. The present invention comprehensively designs multiple sets of primer-probe combinations for these two targets respectively, and screens and optimizes them according to the specificity of the reaction and the amplification efficiency. Finally, the primer and probe combinations of the present invention are selected to achieve the above extremely excellent effects. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the specific test results of the real-time fluorescence quantitative PCR of the present invention; Figure 2Standard curve graph of in vitro transcribed pUC57-E2 RNA standard product of the present invention; Figure 3 Standard curve graph of in vitro transcribed pUC57-NAS5A RNA standard product of the present invention; Figure 4 Sensitivity analysis graph of in vitro transcribed pUC57-E2 RNA standard product of the present invention; Figure 5 Sensitivity analysis graph of in vitro transcribed pUC57-NAS5A RNA standard product of the present invention. Detailed implementation manners

[0020] The principles and features of the present invention are described below in conjunction with embodiments. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are followed. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0021] Example 1: Primer and probe design Sequence alignment was performed based on the CSFV vaccine C (AF091507.1) and classical swine fever virus wild strains (FJ598612.1, AY027673.1, MK691768.1, FJ607779.1, HQ697228.1, KF233961.1) prevalent in China included in GenBank. The differences between the CSFV vaccine C strain and the wild strains can be used to design primers and probes. Specific primers and probes for differentiating the rabbit-attenuated vaccine strain of CSFV C strain and CSFV wild strains were designed in the CSFV E2 and NS5A genes through Primer Premier5 software, as shown in Table 1. The primers and probes were all synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0022] Table 1 Primer and probe sequence list

[0023] Example 2: Detection condition optimization 1. Preparation of RNA standard product According to the E2 gene sequence of the CSFV vaccine strain C (AF091507.1) in GenBank and the NS5A gene sequences of classical swine fever virus wild strains prevalent in China (FJ598612.1, AY027673.1, MK691768.1, FJ607779.1, HQ697228.1, KF233961.1), gene sequences E2 and NS5A were synthesized respectively, cloned into the PUC57 vector, transformed into competent bacterial cells DH5α, plasmids were extracted, and after sequencing verification, the recombinant plasmids were named pUC57-E2 and pUC57-NS5A as dual fluorescence quantitative templates. The purified plasmids pUC57-E2 and pUC57-NS5A were used as templates for in vitro transcription with T7 in vitro transcription reagents to obtain RNA standards.

[0024] 2. Optimization of primer-probe concentration and annealing temperature The matrix method was used to optimize the primer concentration and probe concentration, with the final primer concentration being 200 - 400 nmol / L and the final probe concentration being 200 - 400 nmol / L to explore the optimal concentration; the annealing temperatures were 58°C, 59°C, 60°C, 61°C, 63°C; the number of cycles was 40; the total reaction system was 25 μL, and after determination, the reaction solution and reaction program were obtained.

[0025] Test results: The final concentrations of 2 pairs of primers and probes were 400 nmol / L, and the optimal annealing temperature was 60°C.

[0026] Example 3: Verification of detection effect Based on the primers and probes designed in Example 1 and the detection conditions optimized in Example 2, the present invention provides an optimized detection method, which specifically includes the following steps: (1) Pretreat the sample, and extract nucleic acid using the FineMag magnetic bead method animal virus DNA / RNA rapid extraction kit (product number: YG503-G20, batch number: 202406009) DNA / RNA extraction kit. (2) Use the extracted nucleic acid as a template, prepare an amplification reaction solution according to the reaction system in Table 2, and perform a dual fluorescence quantitative RT-PCR reaction. Set the reaction program for the dual fluorescence quantitative RT-PCR detection method as: 55°C for 15 min, pre-denaturation at 95°C for 2 min, denaturation at 95°C for 5 s, annealing at 60°C for 35 s, 40 cycles. Table 2 Composition of PCR reaction system

[0027] (3) Collect the fluorescence signals of the two channels and make a result determination.

[0028] 1. Specificity experiment The optimized detection method of the present invention is used to detect the nucleic acids of the RNA standard of CSFV vaccine strain C, the RNA standard of classical swine fever virus field strain, and PRRSV, PPV, PRV, PCV2, PCV3, PEDV, TGEV, PoRV, JEV, SIV, SDCOV.

[0029] Figure 1 This is a schematic diagram of the specific test results of real-time fluorescence quantitative PCR of the present invention. As can be seen from the figure, the detections of PRV, PPV, PCV2, PCV3, PEDV, TGEV, PoRV, PRRSV, JEV, SIV, SDCOV and the negative control (ddH 2 O) are all negative, indicating that the method has good specificity and no cross-reaction with common swine virus disease pathogens, and can be applied to clinical detection. The E2 amplification result of CSFV vaccine strain C is positive; the NS5A amplification result of CSFV field strain is positive, indicating that this method can be used to distinguish CSFV vaccine strain C from field strain.

[0030] 2. Drawing of the standard curve of the RNA standard The in vitro transcribed pUC57-E2 and pUC57-NS5A positive RNA standards were serially diluted 10-fold (1×10 2 copies / μL~1×10 6 copies / μL), and fluorescence quantitative PCR detection was performed using the optimized detection method, and the standard curve was automatically generated by the fluorescence quantitative PCR instrument.

[0031] The results are as Figures 2-3 shown. Figure 2 This is the standard curve diagram of the in vitro transcribed pUC57-E2 RNA standard of the present invention. The standard curve corresponding to the in vitro transcribed pUC57-E2 is: Y=-3.513X + 40.821, R2 = 0.998, E% = 92.6; Figure 3 This is the standard curve diagram of the in vitro transcribed pUC57-NAS5A RNA standard of the present invention. The standard curve corresponding to the in vitro transcribed pUC57-NS5A is: Y=-3.482X + 40.604, R2 = 0.997, E% = 93.7; indicating that within the diluted concentration range, there is a good linear relationship between the template amount and the Ct value.

[0032] 3. Sensitivity test The in vitro transcribed pUC57-E2 and pUC57-NS5A positive RNA standards were serially diluted 10-fold (1×10 2 copies / μL~1×10 6 copies / μL), and fluorescence quantitative PCR detection was performed using the optimized detection method to determine the lowest detection limit.

[0033] The results are as Figures 4-5 shown in Figure 4 the sensitivity analysis diagram of the in vitro transcribed pUC57-E2 RNA standard of the present invention. The lowest detection limit for the in vitro transcribed pUC57-E2 positive RNA standard is 11.75 copies / μL; Figure 5 the sensitivity analysis diagram of the in vitro transcribed pUC57-NAS5A RNA standard of the present invention. The lowest detection limit for the in vitro transcribed pUC57-NS5A positive RNA standard is 39.81 copies / μL; indicating that the detection method established in this study has good sensitivity.

[0034] 4. Repeatability experiment The in vitro transcribed pUC57-E2 and pUC57-NS5A positive RNA standards were serially diluted 10-fold (1×10 4 ~1×10 2 copies / μL), and fluorescence quantitative PCR was performed using the optimized detection method. Three replicates were set for each concentration, and three independent repeated experiments were conducted to determine the stability and repeatability of this method.

[0035] The results are shown in Table 3. When the plasmid concentration was between 1×10 4 ~1×10 2 copies / μL, both the within-group coefficient of variation and the between-group coefficient of variation were less than 5%, indicating that this method has good repeatability.

[0036] Table 3 Repeatability results

[0037] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A PCR primer probe set for detecting C strain of classical swine fever virus vaccine and wild strain of classical swine fever virus, characterized in that: The invention comprises specific primers and specific probes; the specific primers comprise specific primers for CSFV E2 gene of C strain of classical swine fever virus vaccine and specific primers for detecting NS5A gene of CSFV wild strain of classical swine fever virus; the specific primers for detecting CSFV E2 gene comprise forward primer CSFV-CF and reverse primer CSFV-CR, the nucleotide sequence of the forward primer CSFV-CF is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer CSFV-CR is shown in SEQ ID NO.2; the specific primers for detecting CSFV NS5A gene comprise forward primer CSFV-WF and reverse primer CSFV-WR, the nucleotide sequence of the forward primer CSFV-WF is shown in SEQ ID NO.4, and the nucleotide sequence of the reverse primer CSFV-WR is shown in SEQ ID NO.5; the specific probes comprise specific probes CSFV-CP for CSFV E2 gene of C strain of classical swine fever virus vaccine and CSFV-CR for detecting NS5A gene of CSFV wild strain of classical swine fever virus. The specific probe of NS5A gene is CSFV-WP, the nucleotide sequence of CSFV-CP is shown in SEQ ID NO.3, and the nucleotide sequence of CSFV-WP is shown in SEQ ID NO.

6.

2. The PCR primer probe set according to claim 1, characterized in that: The 5' ends of the specific probes CSFV-CP and CSFV-WP are labeled with a fluorescent reporter group, and the 3' ends are labeled with a fluorescent quencher group.

3. The PCR primer probe set according to claim 2, characterized in that: The fluorescent reporter group labeled at the 5' end of the specific probe CSFV-CP is FAM, and the fluorescent quencher group labeled at the 3' end is BHQ1.

4. The PCR primer probe set according to claim 2, characterized in that: The fluorescent reporter group labeled at the 5' end of the specific probe CSFV-WP is VIC, and the fluorescent quencher group labeled at the 3' end is BHQ1.

5. A kit for detecting C strain of classical swine fever virus vaccine and wild strain of classical swine fever virus, characterized in that: The kit comprises the PCR primer probe set according to any one of claims 1 to 4.

6. A method for detecting C strain of classical swine fever virus vaccine and wild strain of classical swine fever virus, characterized in that: The following steps are involved: (1) Extracting DNA / RNA from the sample to be tested and using the DNA / RNA of the sample to be tested as a template; (2) preparing an amplification reaction solution using the primer probe set according to any one of claims 1 to 4, and performing a dual fluorescence quantitative RT-PCR reaction; (3) Collect the fluorescence signals of the two channels and make a judgment on the results.

7. The method according to claim 6, characterized in that The amplification reaction solution includes the following components: template, 2×One Step U+Mix, One Step U+Enzyme Mix, forward primer CSFV-CF, reverse primer CSFV-CR, specific probe CSFV-CP, forward primer CSFV-WF, reverse primer CSFV-WR, specific probe CSFV-WP and deionized water.

8. The method according to claim 7, characterized in that The concentrations of the forward primer CSFV-CF, the reverse primer CSFV-CR, the specific probe CSFV-CP, the forward primer CSFV-WF, the reverse primer CSFV-WR and the specific probe CSFV-WP in the amplification reaction solution are all 400 nmol / L.

9. The method according to claim 6, characterized in that The program of the dual fluorescence quantitative RT-PCR reaction is: 55°C for 15 min, 95°C pre-denaturation for 2 min, 95°C denaturation for 5 s, 60°C annealing for 35 s, and 40 cycles.

10. The method according to claim 6, characterized in that The specific determination method of step (3) is as follows: when the E2 gene detects an amplification curve, it is determined to be the C strain of the classical swine fever virus vaccine; when the NS5A gene detects an amplification curve, it is determined to be the wild strain of the classical swine fever virus.

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