FN Archimer Export Format
PT J
TI Development of a reverse transcription-PCR-DNA enzyme immunoassay for detection of "Norwalk-like" viruses and hepatitis A virus in stool and shellfish
BT 
AF SCHWAB, Kellogg
   NEILL, Frederick
   LE GUYADER, Soizick
   ESTES, Mary
   ATMAR, Robert
AS 1:;2:;3:;4:;5:;
FF 1:;2:;3:PDG-DEL-MP-MIC;4:;5:;
C1 Baylor Coll Med, Div Mol Virol, Dept Mol Virol & Microbiol, Houston, TX 77030 USA.
   Baylor Coll Med, Dept Med, Houston, TX 77030 USA.
   IFREMER, Microbiol Lab, F-44311 Nantes, France.
C2 BAYLOR COLL MED, USA
   BAYLOR COLL MED, USA
   IFREMER, FRANCE
SI NANTES
SE PDG-DEL-MP-MIC
IN WOS Ifremer jusqu'en 2018
   copubli-int-hors-europe
IF 3.688
TC 50
UR https://archimer.ifremer.fr/doc/2001/publication-1267.pdf
LA English
DT Article
DE ;Bivalvia;Immuoessay;DNA enzyme;Reverse transcription PCR;Detection  method;Enteric virus
AB Outbreaks of food- and waterborne gastroenteritis are being increasingly reported throughout the world. The analysis of environmental samples by ne,ver diagnostic techniques such as reverse transcription-PCR (RT-PCR) amplification of nucleic acid has begun to identify human enteric viruses (predominantly "Norwalklike" viruses [NLVs]) as the cause of many of these outbreaks. To streamline NLV detection from environmental samples such as shellfish, we have developed an RT-PCR-oligoprobe amplification and detection method using several new procedures that enable confirmed RT-PCR amplification and product detection in 1 day. The new steps include replacing reverse transcriptase and Tag polymerase with rTth polymerase, a heat-stable enzyme that functions as both a reverse transcriptase and DNA polymerase, in a single-tube, single-buffer, elevated temperature reaction. An internal standard Norwalk virus (NV) RNA control is added to each RT-PCR to identify sample inhibition, and thermolabile uracil N-glycosylase is incorporated into the reaction to prevent PCR product carryover contamination. Finally, RT-PCR-generated amplicons are detected in microtiter wells using virus-specific biotinylated oligoprobes in an enzyme-linked immunosorbent assay-based format. The DNA en;cyme immunoassay is based on the capture of PCR product by biotinylated probes fixed onto individual streptavidin-coated wells. Using this method, low levels of NV were detected in stool and both NLV and hepatitis A virus were detected in bivalve mollusks following bioaccumulation. The method also successfully detected NLV in oysters implicated in an outbreak of NLV gastroenteritis. This method dramatically decreases the time needed for analysis and is amenable to automation.
PY 2001
PD FEB
SO Applied and environmental microbiology
SN 0099-2240
PU American society for microbiology
VL 67
IS 2
UT 000166844600033
BP 742
EP 749
DI 10.1128/AEM.67.2.742-749.2001
ID 1267
ER

EF