Departamento de Microbiologia, Facultad de Ciencias, Universidad de Malaga, 29071 Malaga, Spain.
J Fish Dis. 2010 Apr;33(4):311-9. doi: 10.1111/j.1365-2761.2009.01123.x. Epub 2010 Jan 3.
A non-destructive procedure based on nested RT-PCR and dot-blot hybridization has been developed for the detection of asymptomatic IPNV-carrier fish. The pair of primers designed for RT-PCR amplified a 599-bp fragment of the pVP2 region within the polyprotein gene, resulting in the detection of IPNV genotype III.1. The use of a nested RT-PCR allowed the amplification of IPNV genotypes III.1 and I.2. In addition, a 191-bp probe was designed for hybridization studies used in combination with the nested RT-PCR. The application of the nested RT-PCR to analyse blood samples from asymptomatic redbanded seabream, Pagrus auriga, and common seabream, P. pagrus, specimens showed a 53.1% and 77.8% prevalence of IPNV-carriers, respectively. The combination of nested RT-PCR and dot-blot hybridization increased the detection rates up to 100% for redbanded seabream and 94.4% for common seabream. Therefore, the protocol described in this study is highly sensitive and specific for the detection of IPNV in asymptomatic carrier fish, and, in addition, the results demonstrate the carrier state in two newly cultured sparid species in southern Spain.
已开发出一种基于嵌套 RT-PCR 和斑点杂交的非破坏性程序，用于检测无症状 IPNV 携带者鱼类。设计用于 RT-PCR 的引物对扩增多蛋白基因内 pVP2 区域的 599bp 片段，从而检测到 IPNV 基因型 III.1。嵌套 RT-PCR 的使用允许扩增 IPNV 基因型 III.1 和 I.2。此外，还设计了一个 191bp 的探针用于与嵌套 RT-PCR 结合进行杂交研究。将嵌套 RT-PCR 应用于分析无症状红鳍东方鲀和真鲷的血液样本，结果显示 IPNV 携带者的患病率分别为 53.1%和 77.8%。嵌套 RT-PCR 和斑点杂交的组合将红鳍东方鲀的检测率提高到 100%，真鲷的检测率提高到 94.4%。因此，本研究中描述的方案对检测无症状携带鱼类中的 IPNV 具有高度的敏感性和特异性，此外，结果还证明了西班牙南部两种新养殖的鲷科鱼类的携带状态。
