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Polymerase Chain Reaction Detection and Typing of Rotaviruses in Fecal Specimens

  • Vera Gouvea
  • Roger I. Glass
Part of the Frontiers of Virology book series (FRVIROLOGY, volume 1)

Summary

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.

Keywords

Polymerase Chain Reaction Assay Polymerase Chain Reaction Method Fecal Specimen Outer Capsid Protein Polymerase Chain Reaction Typing 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag Berlin Heidelberg 1992

Authors and Affiliations

  • Vera Gouvea
    • 1
  • Roger I. Glass
    • 2
  1. 1.Molecular Biology Branch, Division of MicrobiologyCenter for Food Safety and Applied Nutrition, Food and Drug AdministrationWashington, DCUSA
  2. 2.Viral Gastroenteritis Unit, Division of Viral and Rickettsial DiseasesCenter for Infectious Diseases, Centers for Disease ControlAtlantaUSA

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