Development And Preliminary Application Of Fluorescent Quantitative PCR For Detection Of PCV₂,PRV And PPV In The Semen Of Boars

The DNA viruses, including porcine circovirus type 2 (PCV2), pseudorabies virus (PRV) and porcine parvovirus (PPV), result in reproductive syndromes, and can be communicable via the semen of boarsand hence severely hinder sustainable development of pig industry. Real-time quantitative PCR (RT-qPCR) has been widely used in the detection of pathogens, but it has never been employed in the detection of DNA viruses in swine semen. Quantitative PCR using SYBR Green I fluorescence for PRV and TaqMan fluorescence quantitative PCR for PPV were established in present study to provide simple, convenientand accurate detection for those virues.1. The establishment of SYBR Green I fluorescence quantitative PCR for PRV detectionPRV gene sequences from the GenBank were analysized for conserved region using sequence alignment by Blast, and specific primers for PRV were obtained using Primer Premier 5.0. Subsequently, the recombinant plasmids were constructed and used further as a standard for establishment of the kinetics amplification curve and standard curve of qPCR. The PCV2, PRRSV, JEV, CFSV and PPV positive samples were used for the specificity test for the established method. Results showed that the amplification kinetics curve was linear (R2:0.998, E=103.854%), and the method had high specificity for PRV positive samples as it showed negative result in other samples. Meanwhile, the method could detect as low as 27.9 copies/μL PRV in recombinant plasmids and 16TCIDso/100μL PRV in boar semen samples,more sensitive than the national standard method. The coefficient of variation was found lower than 3.5%. Therefore, the developed method had a good linearity, high sensitivity and repeatability.2. The establishment of TaqMan fluorescence quantitative PCR for PCV2 and PPV detection.PCV2 and PPV gene sequences from the GenBank were analysized for conserved region using sequence alignment by Blast, and specific primers for PCV2 and PPV were obtained using Primer Premier 5.0 and Primer Express 3.0. Subsequently, recombinant plasmids were constructed and used further as the standard for establishment of the kinetics amplification curve and standard curve of qPCR. The PRRSV, JEV, CFSV and PRV positive samples were used for the specificity test. Results showed that the amplification kinetics curves were linear (PCV2:R2=0.999, E=102.061%; PPV:R2=0.998, E=99.112%), and the method had high specificity for PCV2 and PPV positive samples as it showed negative result in other samples.. Meanwhile, the method could detect as low as 10 copies/μL PCV2 in recombinant plasmids and 1TCID50/100μL PRV in boar semen samples,more sensitive than the national standard method. The developed method could detect as low as 13.9 copies/μL PPV in recombinant,more sensitive than the regular PCR. The coefficient of variation was found lower than 3%. Therefore,the developed method had a good linearity, high sensitivity and repeatability.3. The establishment of internal standard TaqMan fluorescence quantitative PCR for PCV2 detection.ORF2 of PCV2 from the GenBank was analysized for conserved region using sequence alignment by Blast, and specific primers for PCV2 were obtained using Primer Premier 5.0 and Primer Express 3.0. Meanwhile, an Internal Amplification Control (IAC) template with bridge-building PCR method was used in the real-time PCR detection system. Subsequently, two recombinant plasmids containing specific primers and IAC were constructed as standards for establishment of the kinetics amplification curve and standard curve of qPCR. The PRV, PRRSV, JEV, CFSV and PPV positive samples were using for the specificity test. Results showed that the amplification kinetics curve was linear (R2:0.991, E=106.0%), and the method had high specificity cause there was no cross reaction with other virues. Meanwhile, the method could detect as low as 100 copies/μL PCV2 in recombinant plasmids, or more than 1TCID50/1mL PCV2. This method showed higher sensitivity and repeatability than simple TaqMan qPCR and GB/T 21674-2008.4. The clinical monitoring of PCV2,PRV and PPV in boar semen using the developed methods109 boar semen samples from 15 farms were collected.Results showed that samples from 14 farms (93.33%,14/15) were positive. Among them, the positive ratio for PCV2 was 51.37%(56/109, viral load:1.71 × 106～1.00 × 103 copies/ml); the positive ratio for PRV was 18.35% (20/109, viral load:1.13 X 104～2.25× 103 copies/ml); and positive ratio for PPV was 28.44%(31/109, viral load:1.84× 105～8.44× 102copies/ml). The ratio of combined infection of the three viruses was 8.26%(9/109), the ratio for mixed infection of PCV2 and PRV was 9.17%(10/109), the ratio for mixed infection of PPV and PCV2 was 12.84%(14/109),and the ratio for mixed infection of PRV and PPV was 14.68% (16/109).These results indicated that there was a widely spread of PCV2,PRV and PPV in pig farms, which should be tookmore attention up on.In conclusion:the established qPCR methods had high sensitivity and specificity,and can be used to monitor the clinic infection of PCV2, PRV and PPV in porcine semen, and also can be used to test the semen quality and provide technical support for the prediction and control of epidemic diseases.