Polymerase Chain Reaction Detection and Typing of Rotaviruses in Fecal Specimens
A two-step polymerase chain reaction (PCR) procedure was developed to identify groups A, B, and C rotavirus and to type human group A strains in fecal extracts. Sample preparation consisted of deproteinization and extraction of viral dsRNA, followed by purification by adsorption to hydroxyapatite. In a first step, viral RNA was reverse transcribed into cDNA copies and amplified by Taq polymerase, using group-specific primers in an individual or in a combined detection assay. The amplified DNAs were then used in a second PCR procedure for confirmation of group B or C rotavirus or identification of human group A serotypes 1-4, 8, and 9. Results were interpreted by examining PCR products after ethidium bromide staining of agarose gels for the lengths characteristic of the expected products. Subpicograms of template RNA were diagnosed directly in human and animal fecal specimens. In addition, large quantities of cDNA suitable for sequencing, cloning, and other techniques of molecular analysis were rapidly obtained from field isolates.
This PCR assay, carried out with the appropriate primers, is applicable to the identification of other rotavirus traits, such as antigenic specificities defined by the vp4 protein (vp4 typing), subgrouping, host specificity, and virulence, as well as low-level identification of rotavirus in specimens of epidemiologic interest.
KeywordsPolymerase Chain Reaction Assay Polymerase Chain Reaction Method Fecal Specimen Outer Capsid Protein Polymerase Chain Reaction Typing
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- Glass RI, Keith J, Nakagomi O, Nakagomi T, Askaa J, Kapikian AZ, Chanock RM, Flores J (1985) Nucleotide sequence of the structural glycoprotein vp7 gene of Nebraska calf diarrhea virus rotavirus: comparison with homologous genes from four strains of human and animal rotaviruses. Virology 141:292–298PubMedCrossRefGoogle Scholar
- Gorziglia M, Hoshino Y, Buckler-White A, Blumentals I, Glass R, Flores J, Kapikian AZ, Chanock RM (1986) Conservation of amino acid sequence of vp8 and cleavage region of 84-kDa outer capsid protein among rotaviruses recovered from asymptomatic neonatal infection. Proc Natl Acad Sci USA 83:7039–7043PubMedCrossRefGoogle Scholar
- Kapikian AZ, Chanock RM (1990) Rotaviruses. In: Fields BN, Knipe DM, Chanock RM, Hirsch MS, Melnick DL, Monath TP, Roizman B (eds) Virology. Raven, New York, pp 1353–1404Google Scholar
- Saif LJ (1989) Non group A rotaviruses. In: Saif LJ, Theil KW (eds) Viral diarrheas of man and animals. CRC Press, Boca Raton, pp 73–95Google Scholar
- Ward RL, McNeal MM, Clemens JD, Sack DA, Rao M, Huda N, Green KY, Kapikian AZ, Coulson BS, Bishop RF, Greenberg HB, Gerna G, Schiff GM (1991) Reactivities of serotyping monoclonal antibodies with culture-adapted human rotaviruses. J Clin Micro-biol 29:449–456Google Scholar