Rapid and sensitive detection of ostreid herpesvirus 1 in oyster samples by real-time PCR.
Herpes and herpes-like virus infections have been reported in various marine mollusc species associated with high mortality rates. Following the characterisation and genome sequencing of ostreid herpesvirus 1 (OsHV-1), specific diagnostic tools have been developed based on conventional PCR techniques or in situ hybridisation. We have now developed a real-time PCR assay for rapid, sensitive and quantitative detection of OsHV-1, and compared it with a conventional PCR technique described previously. The new assay utilised SYBR((R)) Green chemistry with specific primers C(9)/C(10) targeting the C region. The melt curve analysis of OsHV-1 DNA or DNA extracted from infected material showed only one melting temperature peak (75.75+/-0.1 degrees C). The assay had a detection limit of 4 copies/microL of viral genomic DNA and a dynamic range of 5 logs. Using infected oyster samples as template, the assay was about 100-fold more sensitive than single PCR method using C(2)/C(6) primers. The assay was applied successfully for rapid diagnosis (100 min) and quantitation of OsHV-1 in different developmental stages of Crassostrea gigas. Although it already exists a competitive PCR method to quantify OsHV-1 DNA, quantitative data that will emerge in future using the new sensitive and reliable assay will illuminate aspects of pathogenesis, in particular the viral loads in asymptomatic oysters and the kinetics of infection in specific target tissues.
Pepin
J F
JF
Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Laboratoire de Génétique et Pathologie (LGP), 17390 La Tremblade, France. jfpepin@ifremer.fr
Riou
A
A
Renault
T
T
D000818:Animals; D052160:Benzothiazoles; D049896:Crassostrea / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D006564:Herpesviridae / Q000235:genetics / Q000302:isolation & purification*; D006566:Herpesviridae Infections / Q000821:virology*; D009930:Organic Chemicals / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity; D044366:Transition Temperature
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I