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Virologica Sinica, 27 (1) : 1-09, 2012
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
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
 Correspondence: balavirol@gmail.com
(417.73KB)  
Abstract
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.
Received: 30 Nov 2011  Accepted: 3 Sep 2011  Published online: 5 Feb 2012
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