Laboratoire Départemental du Calvados Frank Duncombe, Saint Contest, France.
J Virol Methods. 2010 Dec;170(1-2):86-9. doi: 10.1016/j.jviromet.2010.09.003. Epub 2010 Sep 15.
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).
实时聚合酶链反应（PCR）被认为是一种适合核酸定量的工具，因为它准确、快速且可靠。用于定量太平洋牡蛎（Crassostrea gigas）牡蛎疱疹病毒 1 的参考方案是基于 IFREMER 实验室开发的 Sybr(®) Green 实时 PCR。弗兰克·邓科姆部门实验室开发了一种基于 TaqMan(®)化学的替代方案（替代技术）。参考方法和替代方案的定量限分别为 1000 和 18UG/mg 组织，后者的检测限为 6UG/mg 组织。本研究旨在使用从 210 个幼体中获得的 DNA 样本比较两种方案。根据 Landis 和 Koch 的测量，kappa 指数（0.41）表明两种方案之间存在中度一致性。参考方案阳性的所有样本在替代方案中也为阳性。在参考方案中为阴性的 76 个样本中，有 49 个在替代方案中为阳性。总之，替代方案在灵敏度、特异性和快速性（<3h）方面优于参考方案。
