title: A SYBR green, real-time RT-PCR method to detect and quantitate Norwalk virus in stools.
abstract: A simple, single tube, hot start, real-time reverse transcription-PCR (rt RT-PCR) technique using SYBR green fluorescence was developed for the detection of genogroup I, cluster 1 Norwalk virus (NV) in stools. Sample dilution and heat release of viral RNA was effective as an alternative to more complex procedures to extract viruses from stool specimens. Real-time RT-PCR was applied to 68 stool isolates from patients participating in a NV volunteer study. First derivative melt curves were used to verify NV amplicon and to rule out the presence of primer dimer or spurious product. A dilution end-point standard curve was developed to semi-quantitate minimum virus levels and the results showed the number of RT-PCR amplifiable NV as high as 6.16 x 10(10)g(-1) of stool. The application of these methods was instrumental in identifying three asymptomatic patients who shed viruses in their stools, thus demonstrating a carrier state among seemingly healthy individuals. This study serves as a model for the development of rapid and specific detection, verification, and quantitation procedures for other Noroviruses in stools.
authors.affiliation: Agricultural Research Service, United States Department of Agriculture, James WW Baker Center, Delaware State University, Dover, DE 19901, USA. grichard@desu.edu
authors.forename: Gary P
authors.initials: GP
authors.lastname: Richards
authors.forename: Michael A
authors.initials: MA
authors.lastname: Watson
authors.forename: David H
authors.initials: DH
authors.lastname: Kingsley
mesh_terms: D052160:Benzothiazoles; D017250:Caliciviridae Infections / Q000175:diagnosis*; D002353:Carrier State / Q000175:diagnosis / Q000821:virology; D003959:Diamines; D005243:Feces / Q000821:virology*; D006801:Humans; D009663:Norwalk virus / Q000235:genetics* / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009930:Organic Chemicals; D011804:Quinolines; D012328:RNA Viruses / Q000235:genetics / Q000302:isolation & purification; D012367:RNA, Viral / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I