Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantitation of Japanese encephalitis virus

• Published in: Journal of virological methods Vol. 143; no. 1; pp. 73 - 80
• Main Authors: Santhosh, S.R., Parida, M.M., Dash, P.K., Pateriya, A., Pattnaik, B., Pradhan, H.K., Tripathi, N.K., Ambuj, S., Gupta, N., Saxena, P., Lakshmana Rao, P.V.
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01.07.2007; Amsterdam Elsevier; New York, NY
• Subjects: Biological and medical sciences; Cell Culture Techniques; Encephalitis Virus, Japanese - isolation & purification; Encephalitis, Japanese - cerebrospinal fluid; Encephalitis, Japanese - diagnosis; Encephalitis, Japanese - immunology; Encephalitis, Japanese - virology; Flavivirus; Fundamental and applied biological sciences. Psychology; Humans; India; Japanese encephalitis virus; Microbiology; Organic Chemicals; Real-time PCR; Reverse Transcriptase Polymerase Chain Reaction - methods; Sensitivity and Specificity; SYBR Green I; Techniques used in virology; Virology; India; Real-time PCR; Japanese encephalitis virus; SYBR Green I; Microbiology; Method; Real time; Flavivirus; Virology; Virus; Polymerase chain reaction; Japanese encephalitis group virus; Flaviviridae; Detection; Reverse transcription polymerase chain reaction; Quantitative analysis
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2007.02.011

One-step SYBR Green I-based real-time RT-PCR assay for rapid detection as well as quantitation of Japanese encephalitis virus (JEV) in acute-phase patient CSF samples by targeting the NS3 gene was developed. The assay developed in this study was found to be more sensitive as compared to conventional RT-PCR. The specificity of the reported assay system was established through melting curve analysis as well as by cross-reactivity studies with related members of Flavivirus. The applicability of Real-time PCR assay for clinical diagnosis was validated with 32 suspected acute-phase CSF samples of Gorakhpur epidemic, India, 2005. The improved sensitivity of real-time RT-PCR was reflected by picking up 10 additional samples with low copy number of template in comparison to conventional RT-PCR. The quantitation of the viral load in acute-phase CSF samples was done using a standard curve obtained by plotting cycle threshold ( C t) values versus copy numbers of the RNA template. This is the first report on the application of real-time RT-PCR for detection as well as quantitation of JEV from patient CSF samples. These findings demonstrate the potential clinical application of the reported assay as a sensitive diagnostic test for rapid and real-time detection and quantitation of JEV in acute-phase clinical samples.