Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Atmar, R.L. and Estes, M.K. (2006) The epidemiologic and clinical importance of norovirus infection. Gastroenterol Clin N Am 35, 275–290.
Asanaka, M., Atmar, R.L., Ruvolo, V., Crawford, S.E., Neill, F.H., and Estes, M.K. (2005) Replication and packaging of Norwalk virus RNA in cultured mammalian cells. Proc Natl Acad Sci USA 102, 10327–10332.
Bull, R.A., Tanaka, M.M., and White, P.A. (2007) Norovirus recombination. J Gen Virol 88, 3347–3359.
Hansman, G.S., Oka, T., Katayama, K., and Takeda, N. (2007) Human sapoviruses: genetic diversity, recombination, and classification. Rev Med Virol 17, 133–141.
Atmar, R.L. and Estes, M.K. (2001) Diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses. Clin Microbiol Rev 14, 15–37.
Gray, J.J, Kohli, E., Ruggeri, F.M., Vennema, H., Sánchez-Fauquier, A., Schreier, E., Gallimore, C.I., Iturriza-Gomara, M., Giraudon, H., Pothier, P., Di Bartolo, I., Inglese, N., de Bruin, E., van der Veer, B., Moreno, S., Montero, V., de Llano, M.C., Höhne, M., and Diedrich, S.M. (2007) European multicenter evaluation of commercial enzyme immunoassays for detecting norovirus antigen in fecal samples. Clin Vaccine Immunol 14, 1349–1355.
Anderson, A.D., Heryford, A.G., Sarisky, J.P., Higgins, C., Monroe, S.S., Beard, R.S., Newport, C.M., Cashdollar, J.L., Fout, G.S., Robbins, D.E., Seys, S.A., Musgrave, K.J., Medus, C., Vinjé, J., Bresee, J.S., Mainzer, H.M., and Glass, R.I. (2003) A waterborne outbreak of Norwalk-like virus among snowmobilers-Wyoming, 2001. J Infect Dis 187, 303–306.
da Silva, A.K., Le Saux, J.C., Parnaudeau, S., Pommepuy, M., Elimelech, M., and Le Guyader, F.S. (2007) Evaluation of removal of noroviruses during wastewater treatment, using real-time reverse transcription-PCR: different behaviors of genogroups I and II. Appl Environ Microbiol 73, 7891–7897.
Schwab, K.J., Estes, M.K., Neill, F.H., and Atmar, R.L. (1997) Use of heat release and an internal RNA standard control in reverse transcription-PCR detection of Norwalk virus from stool samples. J Clin Microbiol 35, 511–514.
Boom, R., Sol, C.J., Salimans, M.M., Jansen, C.L., Wertheim-van Dillen, P.M., and van der Noordaa, J. (1990) Rapid and simple method for purification of nucleic acids. J Clin Microbiol 28, 495–503.
Vinje, J., Vennema, H., Maunula, L., von Bonsdorff, C.H., Hoehne, M., Schreier, E., Richards, A., Green, J., Brown, D., Beard, S.S., Monroe, S.S., de Bruin, E., Svensson, L., and Koopmans, M.P.G. (2003) International collaborative study to compare reverse transcriptase PCR assays for detection and genotyping of noroviruses. J Clin Microbiol 41, 1423–1433.
Kageyama, T., Shinohara, M., Uchida, K., Fukushi, S., Hoshino, F.B., Kojima, S., Takai, R., Oka, T., Takeda, N., and Katayama, K. (2004) Coexistence of multiple genotypes, including newly identified genotypes, in outbreaks of gastroenteritis due to Norovirus in Japan. J Clin Microbiol 42, 2988–1995.
Vinje, J. and Koopmans, M.P. (2000) Simultaneous detection and genotyping of “Norwalk-like viruses” by oligonucleotide array in a reverse line blot hybridization format. J Clin Microbiol 38, 2595–2601.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2010 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Atmar, R.L., Neill, F.H., Le Guyader, F.S. (2010). Detection of Human Caliciviruses in Fecal Samples by RT-PCR. In: Stephenson, J., Warnes, A. (eds) Diagnostic Virology Protocols. Methods in Molecular Biology, vol 665. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-817-1_3
Download citation
DOI: https://doi.org/10.1007/978-1-60761-817-1_3
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-60761-816-4
Online ISBN: 978-1-60761-817-1
eBook Packages: Springer Protocols