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J Clin Microbiol. 1991 March; 29(3): 519-523

Detection of group B and C rotaviruses by polymerase chain reaction.

V Gouvea, J R Allen, R I Glass, Z Y Fang, M Bremont, J Cohen, M A McCrae, L J Saif, P Sinarachatanant and E O Caul

Division of Viral and Rickettsial Diseases, Centers for Disease Control, Atlanta, Georgia 30333.

ABSTRACT

We adapted the polymerase chain reaction (PCR) to detect the noncultivatable group B and C rotaviruses and introduced a simple and convenient technique to purify viral RNA from stool specimens. Double-stranded RNA present in stool extracts was purified by adsorption to hydroxyapatite and was used as the template for reverse transcription and polymerase amplification. Primer pairs specific for group B (gene 8) and group C (gene 6) rotaviruses were selected to amplify group-characteristic sizes of cDNA copies readily identifiable in ethidium bromide-stained agarose gels. These primer pairs were used separately in individual PCR assays or were pooled with a primer pair specific for group A rotavirus (gene 9) in a combined PCR assay for the simultaneous detection of all three rotavirus groups. The method was very sensitive and was used to identify both human and porcine strains of group B and C rotaviruses in stool specimens. A second PCR amplification with internal group-specific primers served to increase further the sensitivity of the test and to confirm the diagnostic results obtained in the first amplification.

J Clin Microbiol. 1991 March; 29(3): 519-523




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