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J Virol Methods. 2004 Mar 1;116(1):63-70.

A SYBR green, real-time RT-PCR method to detect and quantitate Norwalk virus in stools.

Author information

1
Agricultural Research Service, United States Department of Agriculture, James WW Baker Center, Delaware State University, Dover, DE 19901, USA. grichard@desu.edu

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.

PMID:
14715308
DOI:
10.1016/j.jviromet.2003.10.011
[Indexed for MEDLINE]

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