Establishment And Application Of A Multiplex Taqman Real-Time RT-PCR Assay For Differential Detection Of North American And European Genotype Prrsv

Porcine reproductive and respiratory syndrome virus (PRRSV) can cause reproductive failure in sows and gilts, and serious respiratory illness in pigs of all ages, especially in piglets. Nowadays, the prevalent PRRSV strains in the fields in China include highly pathogenic PRRSV (HP-PRRSV) and classical PRRSV (C-PRRSV) of North American genotype, as well as PRRSV of European genotype (EU-PRRSV). Furthermore, the prevalent strains are still in the state of constant variance. Therefore, it is very urgent to establish a rapid and specific assay for differential detection of PRRSV.1. Establishment and application of a multiplex TaqMan real-time RT-PCR assay for differential detection of North American genotype PRRSV (NA-PRRSV) and EU-PRRSVA multiplex TaqMan real-time RT-PCR assay was established for differential detection of C-PRRSV, HP-PRRSV and EU-PRRSV The assay utilized two pairs of specific primers and three TaqMan probes which were designed according to the different genomic sequences among C-PRRSV, HP-PRRSV and EU-PRRSV. The assay was highly specific, sensitive and reproducible. The correlation coefficient of the standard curves were over0.999; the assay was highly specific and sensitive with the detection limits of2.68copies/μL for each standard plasmid of C-PRRSV, HP-PRRSV and EU-PRRSV, but no amplification for FMDV, CSFV, PRV, PPV, PCV2and so on. the coefficient of variation (CV) was less than1.5percent for both intra-assay and inter-assay.282clinical samples were detected by the assay and95samples were positive for HP-PRRSV,6samples for C-PRRSV and4samples for both HP-PRRSV and C-PRRSV, while no sample for EU-PRRSV.2. Establishment and application of a multiplex TaqMan real-time RT-PCR assay for differential detection of classical, highly pathogenic and TJM-F92vaccine strains of North American genotype PRRSVThe purpose of this study was to establish one multiplex TaqMan real-time RT-PCR assay to simultaneous and differential detection of C-PRRSV, HP-PRRSV and TJM-F92vaccine strains. The assay utilized two pairs of specific primers and three TaqMan probes which were designed according to the different genomic sequences among C-PRRSV, HP-PRRSV and TJM-F92strain. The assay was highly specific and sensitive with the detection limits of2.68copies/μL for each standard plasmid of C-PRRSV, HP-PRRSV and TJM-F92, but no amplification for FMDV, CSFV, PRV, PPV and PCV2. In addition, the coefficient of variation (CV) was less than1.5percent for both intra-assay and inter-assay. Moreover,324clinical samples were detected by the established assay and6samples for C-PRRSV,105samples were positive for HP-PRRSV,3samples for TJM-F92and4samples for both HP-PRRSV and C-PRRSV.3. Establishment and application of a multiplex TaqMan real-time RT-PCR assay for differential detection of NA-PRRSV and EU-PRRSV and Classical swine feverA multiplex TaqMan real-time RT-PCR assay was established for differential detection of classical swine fever virus (CSFV) and North American (NA) and European (EU) genotype PRRSV. The assay utilized three pairs of specific primers and three TaqMan probes which were designed according to the different genomic sequences among CSFV, NA-PRRSV and EU-PRRSV. The assay could specifically amplify for CSFV, NA-PRRSV and EU-PRRSV, but no amplification for other important porcine pathogens. It was highly sensitive with the detection limit of2.68copies/μL for CSFV, NA-PRRSV and EU-PRRSV, respectively. It had good reproducibility and the coefficient of variation was less than1.5percent for both intra-assay and inter-assay.253clinical samples were detected by the established assay, results showed that20samples were positive for CSFV,86samples for NA-PRRSV,11samples for both CSFV and NA-PRRSV, while none for EU-PRRSV.In conclusion, this study successfully established three methods for differential detection of PRRSV,i.e., a multiplex TaqMan real-time RT-PCR assay for differential detection of NA-PRRSV and EU-PRRSV, a multiplex TaqMan real-time RT-PCR assay for differential detection of C-PRRSV, HP-PRRSV and TJM-F92vaccine strains of NA-PRRSV and a multiplex TaqMan real-time RT-PCR assay for differential detection of NA-PRRSV, EU-PRRSV and CSFV. The results indicated that all these assays could be used as effective tools for rapid detection and epidemic surveillance of PRRSV.