A Rapid and Sensitive One Step-SYBR Green Based Semi Quantitative Real Time RT-PCR for the Detection of peste des petits ruminants Virus in the Clinical Samples / 中国病毒学·英文版

ABSTRACT

A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit(R) for detection and semi-quantitation of peste des petitis ruminants virus (PPRV) using the virus RNA and matrix (M) protein gene-specific primers and compared with established conventional RT-PCR and TaqMan RT-PCR.The assay amplifies a 124 bp fragment of the PPRV M gene with Tm of 78.28 to 78.50.The assay was linear within a range of 50 ng to 0.5 fg total virus RNA with a detection limit (sensitivity) of 0.5 fg.Based on the serial dilution of the live-attenuated PPR vaccine virus,the detection limit was ～0.0001 cell culture infectious dose 50％ units (TCID50).Additionally,swab materials spiked with known titre of vaccine virus were equally well detected in the assay.The standardized rt RT-PCR was easily employed for the detection of PPRV nucleic acid directly in the field and experimental clinical samples.The assay detected the PPRV nucleic acid as early as 3 day post infection (dpi) and up to 20 dpi in swab materials from the experimental samples.The assay was rapid and more sensitive than TaqMan and conventional RT-PCR in the detection of PPRV nucleic acid from the PPR suspected clinical samples of sheep and goats.Therefore,the established,simplified SYBR green rt RT-PCR is an alternative test to the already existing various diagnostic assays and could be useful for rapid clinical diagnosis with advantage in reducing risk of contamination.