Development and Application of a Duplex FQ-PCR for Differential Detection of African Swine Fever Virus and Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus
To develop a specific,sensitive and rapid real-time fluorescence quantitative PCR(FQ-PCR)method for the differential diagnosis of African swine fever virus(ASFV)and highly pathogenic porcine reproductive and respiratory syndrome virus(HP-PRRSV),specific primer/probe pairs were designed for B646L of ASFV and NSP2 of HP-PRRSV for development of a FQ-PCR method based on probe technology.The FQ-PCR method was optimized for its reaction conditions to and verified for its sensitivity,specificity and repeatability.The results showed that the duplex FQ-PCR assay had a good linear relationship at a template range of 10-1-105copies/μL and specifically amplified ASFV and HP-PRRSV genes.The duplex FQ-PCR assay did not amplify the nucleic acid samples of seven pathogens,such as Japanese encephalitis virus(JEV),Classical swine fever virus(CSFV),Porcine parvovirus(PPV),Porcine pseudorabies virus(PRV),Porcine circovirus type 2(PCV2),Porcine reproductive and respiratory syndrome virus(PRRSV)American classic strain VR2332 and healthy porcine spleens.The coefficients of variation of intra-batch and inter-batch tests ranged from 0.53％to 3.14％,indicating good repeatability.The minimum template concentrations for both ASFV and HP-PRRSV were 10 copies/μL.Subsequently,130 clinical samples were tested using this method and the results were compared with the OIE method for ASFV and the national standard method for HP-PRRSV.The results of the duplex FQ-PCR method were consistent with the ASFV OIE method and the HP-PRRSV national standard method.In summary,a duplex FQ-PCR assay was developed in this study to differentiate ASFV from HP-PRRSV,providing a rapid,sensitive and specific detection method for the differential diagnosis of ASFV and HP-PRRSV.
国家科技期刊平台
NSTL
万方数据
维普
