Quantification of hepatitis A virus in shellfish by competitive reverse transcription-PCR with coextraction of standard RNA.
To quantify hepatitis A virus (HAV) in experimentally contaminated mussels, we developed an internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription (RT)-PCR. Deposited directly into the sample, this standard was used both as extraction control and as quantification tool. After coextraction and competitive RT-PCR, standard and wild-type products were detected by differential hybridization with specific probes and a DNA enzyme immunoassay. The quantifiable range with this reproducible method was 10(4) to 10(7) copies of HAV/gram or 400 to 10(6) 50% tissue culture infective doses/ml.
Arnal
C
C
Laboratoire de Virologie, Institut de Biologie, Centre Hospitalier Universitaire, 44093 Nantes Cedex, France. sbi@sante.univ-nantes.fr
Ferre-Aubineau
V
V
Mignotte
B
B
Imbert-Marcille
B M
BM
Billaudel
S
S
D000818:Animals; D001483:Base Sequence; D049872:Bivalvia / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D005517:Foodborne Diseases / Q000517:prevention & control; D006506:Hepatitis A / Q000517:prevention & control; D006507:Hepatovirus / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D006801:Humans; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000235:genetics* / Q000302:isolation & purification* / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012758:Shellfish / Q000821:virology*
D017931:DNA Primers; D012367:RNA, Viral