Detection and quantification of human and bovine noroviruses by a TaqMan RT-PCR assay with a control for inhibition

• DOI: 10.1016/j.mcp.2008.02.003
• Publication Date: 2008-03-07T20:01:13Z
• Authors: Alexandra Scipioni; Isabelle Bourgot; Axel Mauroy; Dominique Ziant; Claude Saegerman; Georges Daube; Etienne Thiry
• Keywords: Staphylococcus aureus; 0303 health sciences; Base Sequence; Transcription, Genetic; Reverse Transcriptase Polymerase Chain Reaction; Molecular Sequence Data; Norovirus; Reference Standards; 3. Good health; Feces; 03 medical and health sciences; Animals; Humans; Cattle; Taq Polymerase; Sequence Alignment

Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and calves. Reverse transcription-polymerase chain reaction (RT-PCR) has become the "gold standard" for detection of noroviruses in faecal and environmental samples. However, false negative results due to co-concentration of RT-PCR inhibitors are a continuous concern. A TaqMan real-time RT-PCR assay making use of a foreign internal RNA control and a RNA standard was developed. Very interestingly, this method is capable of detecting human noroviruses belonging to genogroups I and II, and bovine noroviruses belonging to genogroup III. Inhibitors were removed efficiently by 1/10 dilution of the sample or addition of bovine serum albumin to the RT-PCR mix. This assay was validated with human and bovine stool samples previously tested for norovirus by conventional RT-PCR. The ability to detect norovirus in stool samples that were negative by conventional RT-PCR assay demonstrate the higher sensitivity of the TaqMan assay compared to the conventional RT-PCR assay. This real-time RT-PCR assay allows the detection of both human and bovine noroviruses, avoids false negative results and is able to quantify the level of norovirus contamination.