Send to

Choose Destination
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

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


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.

[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center