Establishment Of Real-time RT-PCR To Detect Different Serotype Of Porcine Reproductive And Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome(PRRS) is clinically characterized by reproductive failure in sows and gilts,pneumonia in grower/finisher pigs,and increased morbidity and mortality in preweaning pigs.The PRRS virus(PRRSV) is a member of the Arteriviridae.The genome is a positive sense,15 kb single strand of RNA consisting of 8 open reading frames(ORFs).Both genetic and antigenic differences have been demonstrated between European and NA PRRS virus.Only NA PRRSV has been spread and reported in China.Real-time fluorescence PCR is characterized by time-saving,easy to handle,decreasing risk of cross-contamination and highly sensitivity and specificity.It is therefore,considered to be a powerful tool for the rapid detection of PRRSV.1.Establishment of Real-time fluorescence RT-PCR assays for detection of North American type porcine reproductive and respiratory syndrome virusTwo pairs of real-time RT-PCR primers and probes,U1 and U6,were designed and synthesized based on the nucleotide sequences of the PRRSV.After optimization of the detection conditions,two methods,U1 and U6 real-time RT-PCR,were developed here for PRRSV detection.The minimal detection limits of U1 and U6 real-time RT-PCR were approximately 10 copies of plasmid DNA.The high specificity of the two methods were demonstrated by detecting 7 viruses,such as Classical swine fever virus(HCV),Pseudorabies virus(PRV),etc.Only PRRSV could be detected.They were considered to be powerful tools for the rapidly detection of PRRSV.2.Establishment of Real-time RT-PCR assays for detection of European type porcine reproductive and respiratory syndrome virusTwo pairs of real-time RT-PCR primers and probes,E1 and E6,were designed and synthesized based on the nucleotide sequences of the PRRSV.After optimization of the detection conditions,two methods,E1 and E6 real-time RT-PCR were developed here for PRRSV detection.The minimal detection limits of E1 and E6 real-time RT-PCR were approximately 616 and 216 copies of plasmid DNA.The high specificity of the two methods were demonstrated by detecting 7 viruses,such as Classical swine fever virus (HCV),Pseudorabies virus(PRV),etc.Only PRRSV could be detected.They can be used for PRRSV inspection and research.