AIMS: To develop and validate assays based on real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for rapid detection and strain identification (European and North American strains) of porcine reproductive and respiratory syndrome virus (PRRSV) by using SYBR Green I and TaqMan probe chemistries. METHODS AND RESULTS: This study describes two alternative assays based on real-time RT-PCR for rapid detection and strain identification of PRRSV in comparison with conventional RT-PCR. The first assay utilized SYBR Green I with melting curve analysis; another assay was performed using strain-specific TaqMan probes. Primers were selected from the conserved regions within ORF7 (N) of both strains whereas two TaqMan probes labelled with different fluorescent dyes were specifically designed for each strain. The result of strain identification was confirmed by direct sequencing. Both assays can be used for rapid detection and strain identification of PRRSV with a sensitivity of 10(4) and 10(3) copies microl(-1) for SYBR Green and TaqMan probe, respectively. CONCLUSIONS: Real-time RT-PCR is a powerful method combining rapidity, specificity and efficiency for large-scale screening and strain identification of PRRSV. SIGNIFICANCE AND IMPACT OF THE STUDY: The data indicate that the methods developed are invaluable for detecting low levels of PRRSV infection in swine.
AIMS: To develop and validate assays based on real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for rapid detection and strain identification (European and North American strains) of porcine reproductive and respiratory syndrome virus (PRRSV) by using SYBR Green I and TaqMan probe chemistries. METHODS AND RESULTS: This study describes two alternative assays based on real-time RT-PCR for rapid detection and strain identification of PRRSV in comparison with conventional RT-PCR. The first assay utilized SYBR Green I with melting curve analysis; another assay was performed using strain-specific TaqMan probes. Primers were selected from the conserved regions within ORF7 (N) of both strains whereas two TaqMan probes labelled with different fluorescent dyes were specifically designed for each strain. The result of strain identification was confirmed by direct sequencing. Both assays can be used for rapid detection and strain identification of PRRSV with a sensitivity of 10(4) and 10(3) copies microl(-1) for SYBR Green and TaqMan probe, respectively. CONCLUSIONS: Real-time RT-PCR is a powerful method combining rapidity, specificity and efficiency for large-scale screening and strain identification of PRRSV. SIGNIFICANCE AND IMPACT OF THE STUDY: The data indicate that the methods developed are invaluable for detecting low levels of PRRSV infection in swine.