title: Development and comparative evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantification of West Nile virus in human patients.
abstract: The recent outbreaks of West Nile Virus (WNV) in the Northeastern American continents and other regions of the world have made it essential to develop an efficient protocol for surveillance of WN virus. Nucleic acid based techniques like, RT-PCR have the advantage of sensitivity, specificity and rapidity. A one step single tube Env gene specific real-time RT-PCR was developed for early and reliable clinical diagnosis of WNV infection in clinical samples. The applicability of this assay for clinical diagnosis was validated with 105 suspected acute-phase serum and plasma samples from the recent epidemic of mysterious fever in Tamil Nadu, India in 2009-10. The comparative evaluation revealed the higher sensitivity of real-time RT-PCR assay by picking up 4 additional samples with low copy number of template in comparison to conventional RT-PCR. All the real-time positive samples further confirmed by CDC reported TaqMan real-time RT-PCR and quantitative real-time RT-PCR assays for the simultaneous detection of WNV lineage 1 and 2 strains. The quantitation of the viral load samples was done using a standard curve. These findings demonstrated that the assay has the potential usefulness for clinical diagnosis due to detection and quantification of WNV in acute-phase patient serum samples.
authors.affiliation: Division of Virology, Defence Research and Development Establishment (DRDE), Jhansi Road, Gwalior 474002, MP, India. Electronic address: jyotishukla2@yahoo.co.in.
authors.forename: Jyoti S
authors.initials: JS
authors.lastname: Kumar
authors.affiliation: Division of Virology, Defence Research and Development Establishment (DRDE), Jhansi Road, Gwalior 474002, MP, India.
authors.forename: Divyasha
authors.initials: D
authors.lastname: Saxena
authors.affiliation: Division of Virology, Defence Research and Development Establishment (DRDE), Jhansi Road, Gwalior 474002, MP, India.
authors.forename: Manmohan
authors.initials: M
authors.lastname: Parida
mesh_terms: D000210:Acute-Phase Reaction / Q000097:blood / Q000175:diagnosis / Q000821:virology; D001483:Base Sequence; D052160:Benzothiazoles; D003959:Diamines; D006801:Humans; D008969:Molecular Sequence Data; D009930:Organic Chemicals; D011804:Quinolines; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D014759:Viral Envelope Proteins / Q000235:genetics; D019562:Viral Load / Q000235:genetics; D014901:West Nile Fever / Q000097:blood / Q000175:diagnosis / Q000821:virology*; D014902:West Nile virus / Q000235:genetics*
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D014759:Viral Envelope Proteins; C098022:SYBR Green I
keywords: CDC TaqMan real-time RT-PCR; Clinical samples; Cycle sequencing; Envelope gene; Real-time RT-PCR; West Nile virus