Evaluation of reverse transcription and polymerase chain reaction (RT/PCR) for the detection of rotaviruses: applications of the assay

Research in Virology
J BuesaJ Prat

Abstract

Our aim was to evaluate the reverse transcription and polymerase chain reaction (RT/PCR) technique for the detection of rotavirus shedding by infected children as a routine diagnostic procedure, in comparison to the enzyme-linked immunosorbent assay (ELISA), electron microscopy (EM) and polyacrylamide gel electrophoresis (PAGE) of rotavirus double-stranded RNA. Two-hundred and twenty stool specimens were collected from infants and young children with diarrhoea, and 10-20% faecal suspensions were made. Several methods of rotavirus dsRNA extraction were assayed. Electrophoretic analysis of viral RNA was carried out on 10% polyacrylamide gels followed by silver staining. RT/PCR was performed using oligonucleotide primers specific for both 3' and 5' ends of the rotavirus gene encoding VP7 which are highly conserved among group A rotaviruses. Following RNA extraction with phenol-chloroform and ethanol precipitation, RT/PCR could detect rotaviral RNA in only 11 of 25 samples known to contain rotaviruses by conventional methods. The purification of RNA extracts by CF11 cellulose and the application of the RNAID method were equally effective in extracting RNA and/or removing inhibitory substances from the faecal samples. RT/PCR led to ...Continue Reading

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Jul 14, 1998·Journal of Medical Virology·G G GonzálezJ E Ludert
May 22, 2002·Journal of Virological Methods·Guillermo Adolfo VillegasGraciela Glikmann
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