| Summary: | An in-house nucleic acid sequence based amplification (NASBA) assay protocol for
the detection of EV-A71 from both virus isolates and clinical specimens was developed in
this study. A primer set, targeting the VP1 gene region, was designed and the sensitivity
and specificity was tested using conventional RT-PCR and subsequently used in a NASBA
assay. The detection of EV-A71 NASBA amplicons was successfully achieved using
ethidium bromide-stained RNA agarose gel electrophoresis. The in-house EV-A71 NASBA
protocol detected a panel of 54 of 60 EV-A71 virus isolates from different genogroups and
subgenogroups and no cross-reactivity was observed against other EV serotypes. The
NASBA assay was additionally evaluated on a panel of, 41 clinical specimens of various
sample types from HFMD cases in Sarawak. Twenty-one (95.5%) of 22 clinical specimens
that were confirmed to be EV-A71 positive by RT-PCR and virus isolation were
successfully detected by the NASBA assay. The other 19 clinical specimens that were
concordantly EV-A71 negative by both RT-PCR and virus isolation, were not detected by
the EV-A71 NASBA assay. Verification of the EV-A71 NASBA amplicons from virus
isolates and clinical specimens were successfully achieved by sequencing of the amplified
region. In conclusion, the NASBA assay developed in this study provides a useful
alternative for the screening and detection of EV-A71.
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