Unidad de Ictiopatología, Departamento de Microbiología y Parasitología, Instituto de Acuicultura, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
J Virol Methods. 2009 Dec;162(1-2):155-62. doi: 10.1016/j.jviromet.2009.07.033. Epub 2009 Aug 7.
Viral haemorrhagic septicaemia virus (VHSV), a member of the Rhabdoviridae family, is a major viral pathogen of cultured salmonid fish, and also infects a wide range of marine fish species. In the present study, two real time PCR protocols (based on SYBR Green and TaqMan) were developed for the detection of strains belonging to all known genotypes of VHSV. Validation of the procedure, in terms of sensitivity, specificity and repeatability/reproducibility (R&R), was also performed. For this purpose, several pairs of primer amplifying regions corresponding to viral G and N genes were assayed. In the SYBR Green-based real time PCR, these primers failed to detect strains from some of the genotypes and/or showed low R&R. In order to improve the detection capacity, a multiplex procedure was designed, which enabled detection of all strains, with high R&R. The sensitivity of the procedure was measured, and a detection limit of 1 fg/microl of viral RNA or 10 copies of cloned plasmid was established. On the other hand, the TaqMan probe-based multiplex real time PCR detected all European strains, with similar levels of sensitivity and R&R, but failed to detect the American types.
病毒性出血性败血症病毒（VHSV）属于 Rhabdoviridae 科，是养殖鲑鱼的主要病毒性病原体，也感染多种海洋鱼类。在本研究中，开发了两种基于实时荧光定量 PCR（SYBR Green 和 TaqMan）的方法，用于检测所有已知 VHSV 基因型的毒株。还对该方法的灵敏度、特异性和重复性/再现性（R&R）进行了验证。为此，对相应的病毒 G 和 N 基因的几个引物扩增区域进行了测试。在基于 SYBR Green 的实时荧光定量 PCR 中，这些引物无法检测到一些基因型的毒株，或者 R&R 较低。为了提高检测能力，设计了一种多重程序，可实现对所有毒株的高 R&R 检测。对该方法的灵敏度进行了测量，确定了病毒 RNA 为 1 fg/µl 或克隆质粒为 10 个拷贝的检测限。另一方面，基于 TaqMan 探针的多重实时荧光定量 PCR 可以检测到所有欧洲株，灵敏度和 R&R 相似，但无法检测到美洲型。
