Detection of human caliciviruses in fecal samples by rt-PCR

Human caliciviruses, noroviruses in particular, are a common cause of gastroenteritis in persons of all age groups. Although both antigen detection and serologic methods for diagnosis of infection with these viruses have been described, the best and most common methods used for diagnosis are molecular assays. Traditional RT-PCR methods are commonly used for diagnosis, but these require the use of a confirmatory test (such as probe hybridization or sequencing of amplicons). More recently, real-time RT-PCR assays have been developed that allow the rapid and accurate identification of caliciviruses in fecal samples. There is no single primer set that allows the detection of all strains within a calicivirus species, and separate primer pairs are generally used to identify strains belonging to different norovirus genogroups. Inhibition of nucleic acid amplification by substances contained within fecal samples is a common problem facing the diagnostician, but protocols to effectively remove the majority of such inhibitors have now been developed. This chapter describes methods for sample collection and processing of fecal specimens for molecular detection of enteric viruses, and it also describes both traditional and real-time RT-PCR assays for norovirus diagnosis.