Detection of enteroviruses in shellfish by fluorogenic polymerase chain reaction integrated with 96-well microplate scanning.
A one-step procedure was developed to confirm viral targets by using a fluorometric 96-well microplate scanner following polymerase chain reaction (PCR). The fluorogenic PCR, integrated with fluorometric scanning, measured the end point fluorescence of viral PCR amplicon/probe hybrids and permitted the use of nonfluorogenic PCR conditions with addition of a Cy3 fluorophore-labeled linear probe for viruses. This linear probe generated higher ratios of viral signal-to-noise than a comparative beacon probe. Detection efficiency with a Cy3/quencher linear probe was comparable with Southern analysis at the level > or = 0.27 plaque-forming units (PFU) of poliovirus/PCR. For the reaction containing < 0.27 PFU, the fluorometric measurements of the first-round PCR viral amplicon were not as sensitive as Southern analysis; however, equivalent sensitivities were achieved with fluorogenic nested PCR. Concentrates of 11 oyster samples exposed to municipal sewage were tested for enteroviruses; the fluorogenic detection correlated 100% with Southern analysis. This method using fluorometric scanning of viral amplicon is simple; it requires neither continuously monitoring equipment nor redesigning PCR primers; and it accurately detects enteroviruses in oyster sample concentrates in less time than classic spectrophotometry or Southern analysis.
Shieh
Y Carol
YC
yshieh@cfsan.fda.gov
Baric
Ralph S
RS
D000818:Animals; D015139:Blotting, Southern; D004279:DNA, Viral / Q000032:analysis; D004785:Environmental Pollutants / Q000032:analysis; D005456:Fluorescent Dyes; D005470:Fluorometry; D010125:Ostreidae / Q000737:chemistry / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012758:Shellfish / Q000032:analysis / Q000821:virology*; D014780:Viruses / Q000737:chemistry*
D004279:DNA, Viral; D004785:Environmental Pollutants; D005456:Fluorescent Dyes