1.Division of Virology, Indian Veterinary Research Institute, Mukteswar 263138, Uttarakhand, India 2.Project Directorate on Animal Disease Monitoring And Surveillance PD-ADMAS, Bangalore 560024, Karnataka, India 3.Animal Health Division, ICAR-NEH Region, Meghalaya 793103, India 4. Indian Veterinary Research Institute, Bangalore 560024, Karnataka, India 5. National Research Centre on Equines, Hisar 125001, Haryana, India
A sensitive and rapid single step real time (rt) RT-PCR was standardized using one-step Brilliant SYBR Green kit? 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.