Development and use of an internal standard for oyster herpesvirus 1 detection by PCR

Oyster samples were examined using a competitive PCR method in order to detect and quantify oyster herpesvirus 1 (OsHV-1) DNA. Quantitation of viral DNA by competitive PCR assay was based on co-amplification of OsHV-1 DNA and a competitor where a known amount of competitor DNA was present in the same reaction mixture. The competitor was engineered so that it differs in length (deletion of 76 base pairs) from the viral DNA. The assay allowed the detection of 1 fg of viral DNA among 0.5 mg of oyster tissues. The method was used to demonstrate the absence of PCR inhibitors in oyster spat ground tissues. PCR inhibition was observed in adult oyster samples when the same tissue preparation procedure was used. On the contrary, classical phenol/chloroform DNA extraction from adult oyster tissues allowed co-amplification of the internal standard competitor and the viral DNA. The method was successfully used to demonstrate the presence of viral DNA in asymptomatic adult oysters. Quantitation of OsHV-1 DNA in infected spat and asymptomatic adult oysters was also carried out. Viral DNA (1.5-325 pg) were detectable in 0.5 mg of oyster tissues in adults. The amounts of viral DNA in infected oyster spat varied from 750 pg to 35 ng per 0.5 mg of ground tissues.