Detection and quantification of human and bovine noroviruses by a TaqMan RT-PCR assay with a control for inhibition.
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.
Scipioni
Alexandra
A
Department of Infectious and Parasitic Diseases, Virology, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium.
Bourgot
Isabelle
I
Mauroy
Axel
A
Ziant
Dominique
D
Saegerman
Claude
C
Daube
Georges
G
Thiry
Etienne
E
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D005243:Feces / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D029322:Norovirus / Q000235:genetics* / Q000302:isolation & purification*; D012015:Reference Standards; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D016415:Sequence Alignment; D013211:Staphylococcus aureus / Q000235:genetics; D019914:Taq Polymerase / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
D019914:Taq Polymerase