Dual fluorescent quantitative PCR (Polymerase Chain Reaction) primer probe combination for identifying pseudorabies virus vaccine strain and wild strain and application of dual fluorescent quantitative PCR primer probe combination

Abstract

The invention discloses a dual fluorescent quantitative PCR (polymerase chain reaction) primer probe combination for identifying a PRV (pseudorabies virus) vaccine strain and a wild strain. The dual fluorescent quantitative PCR primer probe combination comprises two pairs of primers gB-F and gB-R, gE-F and gE-R, and probes gB-probe and gE-probe, wherein the two pairs of primers are used for specifically amplifying a PRV gB gene and a PRV gE gene; through existence of real-time fluorescence PCR amplification curves of the PRV gB gene and the gE gene, a PRV vaccine strain (gE gene deletion) and a wild strain can be rapidly, specifically, sensitively and stably identified, technical support can be provided for porcine pseudorabies epidemic disease monitoring, and thorough purification of the PRV in a live pig breeding link is facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and relates to a dual fluorescence quantitative PCR primer-probe combination for differentiating pseudorabies virus vaccine strains and wild strains and its application. Background Art

[0002] "Pigs are the foundation for ensuring food security for the country", and the pig industry is one of the important pillar industries in the development of animal husbandry. Porcine pseudorabies is an infectious disease caused by the infection of Pseudorabies virus (PRV), which seriously endangers the health of pig herds. Pigs are the source of infection and natural reservoir hosts of PRV. After pigs are infected with PRV, it can cause fever. Breeding pigs mainly show reproductive disorders. Sows can give birth to dead fetuses, mummified fetuses and abortions. It is most harmful to piglets. Piglets can show neurological symptoms and the mortality rate is as high as 100%; the harm to adult pigs is not serious, generally latent infection. Medium and large pigs will show respiratory symptoms and the overall production performance will deteriorate. Porcine pseudorabies is prevalent in almost all countries and regions in the world, causing huge economic losses to the global pig breeding industry.

[0003] In the "National Medium- and Long-Term Animal Disease Prevention and Control Plan (2012-2020)", porcine pseudorabies was included in the list of diseases to be eradicated. The importance of porcine pseudorabies eradication was also clarified in the "Notice on Carrying out the Evaluation and Certification of the Eradication of Major Animal Diseases in Large-scale Farms" issued by the China Animal Disease Prevention and Control Center.

[0004] At present, the prevention and control of porcine pseudorabies in clinical practice mainly focuses on the prevention of immunization with attenuated live vaccines. Since gE is an important virulence gene for PRV neurological infection, deletion of this gene can significantly reduce the pathogenicity of PRV while retaining its immunogenicity and infectivity. Therefore, most of the attenuated live vaccines widely used in the market at present are mainly characterized by the deletion of the gE gene, such as the commonly used Bartha-K61 vaccine in clinical practice. Summary of the Invention

[0005] In order to quickly, specifically, sensitively and stably differentiate PRV vaccine strains (gE gene deletion) and wild strains, provide technical support for the monitoring of porcine pseudorabies, and contribute to the complete eradication of PRV in the pig breeding link, the purpose of the present invention is to provide a dual fluorescence quantitative PCR primer-probe combination for differentiating PRV vaccine strains and wild strains and its application.

[0006] Through research, the present invention provides the following technical solutions:

[0007] 1. A dual fluorescence quantitative PCR primer-probe combination for differentiating PRV vaccine strains and wild strains, including a primer pair gB-F and gB-R for specifically amplifying the PRV gB gene, a primer pair gE-F and gE-R for specifically amplifying the PRV gE gene, and probes gB-probe and gE-probe;

[0008] The sequences of the primers are as follows:

[0009] gB-F: 5’-GAGGGGATCGCCGTGCTCTT-3’;

[0010] gB-R: 5’-TCCGTGAAGCGGTTCGTGAT-3’;

[0011] gE-F: 5’-GAGTTCAGCAGCGACGAG-3’;

[0012] gE-R: 5’-CGCCATAGTTGGGTCCATT-3’;

[0013] The sequences of the probes are as follows:

[0014] gB-probe: 5’-VIC-CTACAAGAACGTCATCGTCACGACCG-3’;

[0015] gE-probe: 5’-CGTCACTTCCGGTTTCTCCGGATC-3’;

[0016] The 5’ end of the probe is linked with a reporter fluorophore, and the 3’ end is linked with a quenching group.

[0017] Furthermore, the reporter fluorophore is VIC or FAM, and the quenching group is BHQ1.

[0018] 2. Application of the dual fluorescence quantitative PCR primer-probe combination for differentiating PRV vaccine strains and wild strains in preparing a kit for differentiating PRV vaccine strains and wild strains.

[0019] 3. A kit for differentiating PRV vaccine strains and wild strains, including the dual fluorescence quantitative PCR primer-probe combination for differentiating PRV vaccine strains and wild strains.

[0020] Furthermore, the kit also includes the reagent Premix Ex Taq(Probe qPCR)(2×Conc.).

[0021] 4. A method for differentiating PRV vaccine strains and wild strains using the dual fluorescence quantitative PCR primer-probe combination or the kit for non-diagnostic purposes, comprising the following steps: obtaining the DNA of the sample to be detected, using the obtained DNA as a template, and performing a dual fluorescence quantitative PCR reaction in one reaction system using two pairs of primers gB-F and gB-R, gE-F and gE-R, and probes gB-probe and gE-probe; when both the gB gene and the gE gene are amplified simultaneously, determining that the sample to be detected is a PRV wild strain; when only the gB gene is amplified, determining that the sample to be detected is a PRV vaccine strain.

[0022] Furthermore, the PCR reaction system is as follows: Premix Ex Taq (Probe qPCR) (2×Conc.) 12.5 μL, the final concentrations of gB-F and gB-R are both 2 pmol / μL, the final concentrations of gE-F and gE-R are both 3 pmol / μL, the final concentrations of gB-probe and gE-probe are both 10 pmol / μL, 1 μL of the template, and finally made up to a total volume of 25 μL with water.

[0023] Furthermore, the PCR reaction program is as follows: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, annealing at 60°C for 30 s, 40 cycles (fluorescence signals are collected during the annealing stage).

[0024] The beneficial effects of the present invention are as follows: The present invention provides a dual fluorescence quantitative PCR primer-probe combination for differentiating PRV vaccine strains and wild strains. Through the presence or absence of the real-time fluorescence PCR amplification curves of the PRV gB gene and gE gene, it can quickly, specifically, sensitively, and stably differentiate PRV vaccine strains (gE gene deletion) and wild strains, which can provide technical support for the monitoring of porcine pseudorabies disease and contribute to the thorough purification of PRV in the live pig breeding link. Description of the Drawings

[0025] Figure 1 It is the bacterial liquid PCR verification of PRV gB and PRV gE recombinant plasmids, where lanes 1-5 are the PCR identification results of 5 randomly selected single colonies containing positive recombinant plasmids respectively.

[0026] Figure 2 It is the sensitivity experiment (left) and standard curve (right) of PRV gE single fluorescence quantitative PCR.

[0027] Figure 3 It is the sensitivity experiment (left) and standard curve (right) of PRV gB single fluorescence quantitative PCR.

[0028] Figure 4 It is the sensitivity experiment of PRV gB and PRV gE ordinary PCR.

[0029] Figure 5 For the sensitivity experiment (left) and standard curve (right) of dual fluorescence quantitative PCR of PRV gB and PRV gE.

[0030] Figure 6 For the specificity experiment of dual fluorescence quantitative PCR of PRV gB and PRV gE, where 1 is the PRV genome, and 2 - 4 are the genomes of porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and porcine circovirus type 3 (PCV3) respectively, and 5 is the negative control ddH2O.

[0031] The above Figures 2 to 5 Among them, 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 respectively represent using recombinant plasmid standards pMD - 19T - gB and / or pMD - 19T - gE at 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 copies / μL as templates, and NC represents the negative control ddH2O. Detailed implementation manners

[0032] In order to make the purpose, technical solutions and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below.

[0033] 1. Design of specific primers and probes for PRV gB and PRV gE

[0034] The specific primers and probes for PRV gB and PRV gE were designed and synthesized with reference to the PRV gene sequence (GenBank: KP257591.1) and the PRV gE gene sequence (GenBank: OR062216.1). The fluorescence labels of the PRV gB and PRV gE probes were respectively selected as VIC and FAM as the reporting luminescent groups, and the quenching group was BHQ1 for both (Table 1). The respective primers of PRV gB and PRV gE can amplify target fragments of 133bp and 124bp respectively in PRV positive samples.

[0035] Table 1 Primers and probes required for fluorescence quantitative detection of PRV gB and PRV gE

[0036]

[0037] 2. Preparation of Standard Products of Recombinant Plasmids of PRV gB and PRV gE

[0038] Use the virus genomic DNA / RNA extraction kit (DP315) from Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the DNA of PRV variant JS-2012 as a template. Use the two pairs of primers gB-F and gB-R, gE-F and gE-R in Table 1 and the reaction system and reaction program described in Table 2 for PCR amplification. After the amplified products are identified by 1% agarose gel electrophoresis to have the correct band sizes, cut the gel and recover to obtain the target fragments PRV gB and PRV gE with lengths of 133 bp and 124 bp respectively.

[0039] Table 2 Amplification System of Target Fragments PRV gB and PRV gE

[0040]

[0041] Ligate the obtained target fragments PRV gB and PRV gE with plasmid pMD-19T respectively, then transform them into Escherichia coli DH5α for amplification. Then randomly select 5 single colonies containing recombinant positive plasmids for PCR identification ( Figure 1 ), and sequence verification. The bacteria containing the recombinant positive plasmids with correct verification are cultured on a large scale and the plasmids are extracted. Finally, the recombinant plasmids pMD-19T-gB and pMD-19T-gE containing the target fragments PRV gB and PRV gE are obtained respectively. Extract the above recombinant plasmids and measure their concentrations (ng / μL), and calculate the copy number of the recombinant plasmids according to the following formula: copy number / μL = plasmid concentration (ng / μL) × 10 -9 × 6.02 × 10 23 / 660 / plasmid length (bp). A series of recombinant plasmid standard products are obtained by 10-fold serial dilution of the recombinant plasmids with known copy numbers.

[0042] 3. Optimization of the Single-Fluorescence Quantitative PCR Reaction System for PRV gB and PRV gE

[0043] Use the single-fluorescence quantitative PCR reaction systems corresponding to PRV gB and PRV gE for amplification respectively: Premix ExTaq (Probe qPCR) (2×Conc.) 12.5 μL, with final concentrations of 10 5 、10 4 、10 3The recombinant plasmid standard with 100 copies / μL was used as a template, and the final concentration of the primers was set to three gradients (2.5pmol / μL, 3.5pmol / μL and 4.5pmol / μL). The final concentration of the probe was 10pmol / μL, and the total volume was finally supplemented with ddH2O to 25μL. The reaction conditions of fluorescence quantitative PCR were set on the Bio-rad fluorescence quantitative PCR instrument as follows: 95℃ pre-denaturation for 30s, 95℃ denaturation for 5s, 60℃ annealing for 30s, and 40 cycles (fluorescence signal was collected during the annealing stage). The final concentrations of primers for PRV gB and PRV gE were screened with the minimum number of cycles (Ct value) required to reach the detection threshold of the fluorescence quantitative PCR instrument and the highest fluorescence intensity of the amplification curve as the screening criteria. It was finally determined that in single-plex fluorescence quantitative PCR, the most suitable final concentrations of primers for PRVgB and PRV gE were 2.5pmol / μL and 3.5pmol / μL, respectively.

[0044] 4. Sensitivity test and establishment of standard curve of single-plex fluorescence quantitative PCR method for PRV gB and PRV gE

[0045] The final concentration was 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 The recombinant plasmid standard with 100 copies / μL was used as a template, and ddH2O was set as a negative control. The amplification was performed according to the above-mentioned optimized PRV gB and PRV gE single-plex fluorescence quantitative PCR reaction system and reaction conditions, and the amplification curve and standard curve were generated using the software provided by the real-time fluorescence quantitative PCR instrument. The results are shown in Figures 2 to 4 The single-plex fluorescence quantitative PCR corresponding to PRV gB and PRV gE can detect as low as 10 1 The viral load of 100 copies / μL was 2 orders of magnitude higher than that of the conventional PCR method; the standard curves of PRV gB and PRV gE both had good linear relationships, with a correlation coefficient of R 2 The values were 0.996 and 0.997, the slopes were -2.95 and -3.08, the intercepts were 41.91 and 41.34, and the amplification efficiencies were 118.07% and 110.96%, respectively.

[0046] 5. Sensitivity test and establishment of standard curve of PRV gB and PRV gE dual fluorescence quantitative PCR method

[0047] Referring to the above single fluorescence quantitative PCR method of PRV gB and PRV gE, a double fluorescence quantitative PCR was performed with the two mixed: 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 The recombinant plasmid standards pMD-19T-gB and pMD-19T-gE with 10 copies / μL were used as templates, and ddH2O was set as a negative control. The final concentration of PRV gB primer was 2pmol / μL, and the final concentration of PRV gE primer was 3pmol / μL. The amplification curve and standard curve of the reaction were generated using the software provided by the real-time fluorescence quantitative PCR instrument. The results are shown in Figure 5 The PRV gE and PRV gB dual fluorescence quantitative PCR method has high sensitivity, and the detection limits of PRV gB and PRV gE are as low as 10 1 copies / μL; the linear relationship between the PRV gB and PRV gE standard curves was good, and the correlation coefficient R 2 The values were 0.995 and 0.998, the slopes were -3.08 and -3.16, the intercepts were 40.43 and 40.42, and the amplification efficiencies were 111.27% and 107.30%, respectively.

[0048] 6. Specificity experiment of PRV gB and PRV gE dual fluorescence quantitative PCR method

[0049] The genomes of other common infectious agents with high co-infection rates in farms and similar clinical symptoms, including porcine reproductive and respiratory syndrome (PRRSV), porcine circovirus 2 (PCV2), and porcine circovirus 3 (PCV3), were used as templates. The genome of the wild-type PRV strain was used as a positive control, and ddH2O was used as a negative control to evaluate the specificity of the constructed PRV gB and PRVgE dual fluorescence quantitative PCR method. The results are shown in Figure 6 The gB and gE genes of PRV positive samples had significant PCR amplification curves and smaller Ct values, while the gB and gE genes of other pathogens had no PCR amplification curves, indicating that the established PRV gB and PRV gE dual fluorescence quantitative PCR method has strong specificity.

[0050] 7. Repeatability experiment of PRV gB and PRV gE dual fluorescence quantitative PCR method

[0051] With a concentration of 10 6 , 10 5 , 10 4The recombinant plasmid standards pMD-19T-gB and pMD-19T-gE at copies / μL were used as templates, and intra-assay and inter-assay repeatability experiments were performed 3 times respectively to evaluate the repeatability of the established dual fluorescence quantitative PCR method for PRV gB and PRV gE. The results are shown in Table 3 and Figure 6 , and the coefficient of variation (CV) of intra-assay and inter-assay repeats was below 0.5%, indicating that the established dual fluorescence quantitative PCR method for PRV gB and PRV gE had good repeatability.

[0052] Table 3 Repeatability experiment of the dual fluorescence quantitative PCR method for PRV gB and PRV gE

[0053]

[0054]

[0055] 8. Application of the dual fluorescence quantitative PCR method for PRV gB and PRV gE

[0056] Nasal swab samples were collected from pigs in multiple farms in a certain area. RNA was extracted and reverse transcribed from the 46 collected samples as template samples. Dual fluorescence quantitative PCR reactions were performed in one reaction system using the primer pairs gB-F and gB-R, gE-F and gE-R, and the probes gB-probe and gE-probe. The reaction system was: Premix Ex Taq(Probe qPCR)(2×Conc.) 12.5 μL, the final concentrations of gB-F and gB-R were both 2 pmol / μL, the final concentrations of gE-F and gE-R were both 3 pmol / μL, the final concentrations of gB-probe and gE-probe were both 10 pmol / μL, 1 μL of template, and finally made up to a total volume of 25 μL with ddH2O. The reaction procedure was: pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, annealing at 60°C for 30 s, 40 cycles (fluorescence signals were collected during the annealing stage). When both the gB gene and the gE gene were amplified by PCR, the sample to be detected was determined to be a wild strain of PRV; when only the gB gene was amplified by PCR, the sample to be detected was determined to be a vaccine strain of PRV.

[0057] The results are shown in Table 6. Among the 46 samples, 1 was positive for both PRV gB and PRV gE, 7 were positive for PRV gB but negative for PRV gE, 38 were negative for both PRV gB and PRV gE. The positive rate of the wild strain of PRV was 2.2%, and the positive rate of the vaccine strain of PRV was 15.2%.

[0058] Table 6 Sample detection results

[0059] <![CDATA[gB + gE + > <![CDATA[gB + gE - > <![CDATA[gB - gE - > Total Quantity 1 7 38 46 Percentage 2.2％ 15.2％ 82.6％ 100％

[0060] The above experimental results indicate that the primer pairs gB-F and gB-R, gE-F and gE-R, and the probes gB-probe and gE-probe designed in the present invention can be used to distinguish between PRV vaccine strains and wild strains. The above primer-probe combinations can be made into a kit for distinguishing PRV vaccine strains and wild strains alone or together with other reagents (such as Premix Ex Taq (Probe qPCR) (2×Conc.)). The dual fluorescence quantitative PCR method for PRV gB and PRV gE established using the above primer-probe combinations can be used to distinguish PRV vaccine strains and wild strains for non-diagnostic purposes, and has the advantages of high sensitivity, strong specificity, good repeatability, simplicity and rapidity.

[0061] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A dual fluorescence quantitative PCR primer-probe combination for differentiating pseudorabies virus vaccine strains and wild strains, characterized in that: It includes primer pairs gB-F and gB-R for specifically amplifying the gB gene of pseudorabies virus, primer pairs gE-F and gE-R for specifically amplifying the gE gene of pseudorabies virus, and probes gB-probe and gE-probe; The sequences of the primers are as follows: gB-F: 5’-GAGGGGATCGCCGTGCTCTT-3’; gB-R: 5’-TCCGTGAAGCGGTTCGTGAT-3’; gE-F: 5’-GAGTTCAGCAGCGACGAG-3’; gE-R: 5’-CGCCATAGTTGGGTCCATT-3’; The sequences of the probes are as follows: gB-probe: 5’-VIC-CTACAAGAACGTCATCGTCACGACCG-3’; gE-probe: 5’-CGTCACTTCCGGTTTCTCCGGATC-3’; The 5’ end of the probe is linked with a reporter fluorophore, and the 3’ end is linked with a quenching group.

2. The dual fluorescence quantitative PCR primer-probe combination for differentiating pseudorabies virus vaccine strains and wild strains according to claim 1, wherein: The reporter fluorophore is VIC or FAM, and the quenching group is BHQ1.

3. Use of the dual fluorescence quantitative PCR primer-probe combination for differentiating pseudorabies virus vaccine strains and wild strains as claimed in claim 1 or 2 in the preparation of a kit for differentiating pseudorabies virus vaccine strains and wild strains.

4. Kit for differentiating pseudorabies virus vaccine strains from wild strains, characterized in that, It includes the dual fluorescence quantitative PCR primer-probe combination for differentiating pseudorabies virus vaccine strains and wild strains as claimed in claim 1 or 2.

5. The kit for differentiating a pseudorabies virus vaccine strain and a wild strain as claimed in claim 4, wherein It also includes the reagent Premix Ex Taq(Probe qPCR)(2×Conc.).

6. A method for differentiating a pseudorabies virus vaccine strain from a wild strain using the dual-fluorescent quantitative PCR primer-probe combination according to claim 1 or 2, or the kit according to claim 4 or 5, for non-diagnostic purposes, characterized in that, It includes the following steps: Obtain the DNA of the sample to be detected. Using the obtained DNA as a template, perform a dual fluorescence quantitative PCR reaction in one reaction system with two pairs of primers gB-F and gB-R, gE-F and gE-R, and probes gB-probe and gE-probe; when both the gB gene and the gE gene are amplified simultaneously, determine that the sample to be detected is a wild strain of pseudorabies virus; when only the gB gene is amplified, determine that the sample to be detected is a vaccine strain of pseudorabies virus.

7. The method for differentiating a pseudorabies virus vaccine strain from a wild strain for non-diagnostic purposes as claimed in claim 6, wherein The PCR reaction system is: 12.5 μL of Premix Ex Taq(Probe qPCR)(2×Conc.), the final concentrations of gB-F and gB-R are both 2 pmol / μL, the final concentrations of gE-F and gE-R are both 3 pmol / μL, the final concentrations of gB-probe and gE-probe are both 10 pmol / μL, 1 μL of template, and finally make up to a total volume of 25 μL with water.

8. The method for differentiating a pseudorabies virus vaccine strain from a wild strain for non-diagnostic purposes as claimed in claim 6, wherein, The PCR reaction program is: pre-denaturation at 95 °C for 30 s, denaturation at 95 °C for 5 s, annealing at 60 °C for 30 s, for 40 cycles.

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