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

• Published in: Virologica Sinica 2012-02, Vol.27 (1), p.1-9
• Main Authors: Balamurugan, Vinayagamurthy, Sen, Arnab, Venkatesan, Gnanavel, Yadav, Vinita, Bhanot, Vandna, Bhanuprakash, Veerakyathappa, Singh, Raj Kumar
• Format: Article
• Language: English
• Subjects: Animals; Biochemistry; Biomedical and Life Sciences; Biomedicine; Cell culture; Contamination; detection limit; DNA Primers - genetics; genes; Goats; M gene; Medical Microbiology; Microbial Genetics and Genomics; Microbiology; Molecular Diagnostic Techniques - methods; Molecular Diagnostic Techniques - standards; Oncology; Organic Chemicals - metabolism; Peste des petits ruminants; Peste-des-Petits-Ruminants - diagnosis; Peste-des-petits-ruminants virus - genetics; Peste-des-petits-ruminants virus - isolation & purification; Polymerase chain reaction; Quantitation; Real-Time Polymerase Chain Reaction - methods; Real-Time Polymerase Chain Reaction - standards; Ribonucleic acid; risk; RNA; RNA viruses; RNA, Viral - genetics; Ruminantia; Sensitivity and Specificity; Sheep; Staining and Labeling - methods; Time Factors; Vaccines; Veterinary Medicine - methods; Veterinary Medicine - standards; Viral Matrix Proteins - genetics; Virology; Virology - methods; Virology - standards; viruses
• ISSN: 1674-0769; 1995-820X
• DOI: 10.1007/s12250-012-3219-z

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.