Comparison of two real-time PCR methods for detection of ostreid herpesvirus 1 in the Pacific oyster Crassostrea gigas.
The real-time polymerase chain reaction (PCR) is considered to be a suitable tool for nucleic acid quantitation because it is accurate, rapid and reliable. The reference protocol for quantitation of ostreid herpesvirus 1 in Pacific oysters Crassostrea gigas is based on a Sybr(®) Green real-time PCR developed by the IFREMER laboratory. The Frank Duncombe Departmental Laboratory has developed an alternative protocol based on TaqMan(®) chemistry (alternative technique). The quantitation limits were 1000 and 18UG/mg of tissues for the reference method and alternative protocols, respectively, and the latter protocol has a detection limit of 6UG/mg of tissues. The aim of this study was to compare the two protocols using DNA samples obtained from 210 spat. The kappa index (0.41) indicated a moderate concordance between the protocols, according to the measures of Landis and Koch. All samples that were positive by the reference protocol were also positive by the alternative protocol. Of the 76 samples that were negative by the reference protocol, 49 were positives by the alternative protocol. In conclusion, the alternative protocol is an improvement of the reference protocol in terms of sensitivity, specificity and rapidity (<3h).
Martenot
C
C
Laboratoire Départemental du Calvados Frank Duncombe, Saint Contest, France.
Oden
E
E
Travaillé
E
E
Malas
J P
JP
Houssin
M
M
D000818:Animals; D052160:Benzothiazoles; D049896:Crassostrea / Q000821:virology*; D017931:DNA Primers; D004267:DNA Viruses / Q000235:genetics / Q000302:isolation & purification*; D003959:Diamines; D005188:False Negative Reactions; D020869:Gene Expression Profiling; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I