Establishment And Application Of TaqMan RT-PCR In Detection Of PRRSV (FJ06 Strain) And Its Distributions In The Challenged Pigs

To establish a rapid and exact diagnostic method of PRRSV(FJ06 strain) with TaqMan probe technology and fluorescent RT-PCR;To determine the distributions of PRRSV(FJ06 strain) in differant tissues of pigs challenged with PRRSV(FJ06 strain) with the established method and to provide a scientific informations of epidemiology and pathogenesis of the highly pathogenic PRRSV.Cloning the gene of PRRSV(FJ06 strain) NSP2:According to gene sequences of the NSP2 of the highly pathogenic PRRSV published in GenBank,a pair of primers were designed and synthesized.The strain of PRRSV(PRRSV FJ06),which was provided by our laboratory,was selected for this study.Gene fragment of NSP2 was amplified by RT-PCR and was linked to the PMD-18T vector,actualizing the transformation of DH5α.The results showed that a standard product of the recombinant plasmid was achieved.Establishment of detection method of PRRSV(FJ06 strain) with TaqMan probe technology and fluorescent RT-PCR:According to gene sequences of the highly pathogenic PRRSV published in GenBank,a pair of specific primers and TaqMan probe were were designed and synthesized.A template of PMD-NSP2 was formed and reaction conditions were optimized.The standard plasmid of the PMD-NSP2 was amplified with fluorescent RT-PCR in quantitative series of 10-fold dilution and standard curves were developed.The results showed that the detection method of highly pathogenic PRRSV was established with fluorescent RT-PCR.Sensitivity was up to 6.24×10~2 copies/μL, 10 times higher than conventional PCR.No cross-reacts are observed among PRRSV, PCV2,PRV,HCV,TGEV.Quantitative determination of PRRSV(FJ06 strain) in different tissues of pigs challenged with PRRSV(FJ06 strain):Quantitative determination of PRRSV(FJ06 strain) in different tissues of pigs challenged with PRRSV(FJ06 strain) was carried out with the established method at the 14th day of challange.14 tissue samples were collected.The results showed that all the samples were invaded by the virus,but concentration of virus was higher in heart,lungs,lymph nodes,tonsils,brain and blood than other tissues.No positive result was obtained in the control pigs.Conclusion:1),Mensuration of PRRSV(FJ06 strain) was established with fluorescent RT-PCR.The method is accurate,rapid and efficient.2),Distributions of PRRSV(FJ06 strain) in different tissues of challenged pigs elucidated what tissues were infected easily by the virus and provide a scientific informations of epidemiology and pathogenesis of the PRRSV(FJ06 strain).