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.
KeywordsCellulose Sucrose Glycerol DMSO Chloroform
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