title: 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.
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 T(m) 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 (TCID(50)). 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.
authors.affiliation: Division of Virology, Indian Veterinary Research Institute, Mukteswar 263138, Uttarakhand, India. balavirol@gmail.com
authors.forename: Vinayagamurthy
authors.initials: V
authors.lastname: Balamurugan
authors.forename: Arnab
authors.initials: A
authors.lastname: Sen
authors.forename: Gnanavel
authors.initials: G
authors.lastname: Venkatesan
authors.forename: Vinita
authors.initials: V
authors.lastname: Yadav
authors.forename: Vandna
authors.initials: V
authors.lastname: Bhanot
authors.forename: Veerakyathappa
authors.initials: V
authors.lastname: Bhanuprakash
authors.forename: Raj Kumar
authors.initials: RK
authors.lastname: Singh
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D006041:Goats; D025202:Molecular Diagnostic Techniques / Q000379:methods* / Q000592:standards; D009930:Organic Chemicals / Q000378:metabolism; D029021:Peste-des-Petits-Ruminants / Q000175:diagnosis*; D018107:Peste-des-petits-ruminants virus / Q000235:genetics / Q000302:isolation & purification*; D011804:Quinolines; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D012756:Sheep; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D014730:Veterinary Medicine / Q000379:methods* / Q000592:standards; D014763:Viral Matrix Proteins / Q000235:genetics; D014773:Virology / Q000379:methods* / Q000592:standards
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; D014763:Viral Matrix Proteins; C098022:SYBR Green I