Worldwide, Rv is the major viral agent of severe diarrhea in children and the current focus of intense research to develop an effective vaccine. To evaluate RT-PCR as a means of identifying each gene 9 type in Rv isolates from Korean children
with
diarrhea, Rv gene segment coding for the major outer capsid glycoprotein VP7 was amplified directly from 22 stool specimens and tissue cultures infected with 6 known reference human Rv serotypes(1, 2, 3, 4, 8, 9). Rv double-stranded RNAs(dsRNAs)
extracted from the stool samples and tissue cultures were used as the template for RT, which was followed immediately and in the same reaction mix with amplication, using the Taq polymerase and 6 type-specific primers derived from distinct
regions
on
the gene. The RT-PCR reliably identified the gene 9 types of the 6 reference strains and 10 Rv that had previously been serotyped by an enzyme immunoassay with monoclonal antibodies(Mab-EIA). In addition, this method types 7 (58%) of the 12
stools
nonserotypable by the Mab-EIA. This method, in conjuction with the Mab-EIA typing, did permit a more complete characterization of neutralization genes and antigens of epidemiologically important (vaccine-related interest)Rv. It also provides a
rapid and
efficient means of obtaining large quantities of cDNA suitable for sequencing, cloning, and other genetic studies, precluding the need for cell culture and virus purification.
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