| Summary: | 碩士 === 中臺科技大學 === 生物科技研究所 === 102 === Koi herpesvirus (KHV), also known as Cyprinid herpesvirus 3, is a member of the Alloherpesviridae family. KHV causes high mortality in carp (Cyprinus carpio carpio) and koi (Cyprinus carpio koi). Currently, polymerase chain reaction (PCR) is one of the most commonly used molecular detection method. However, requiring expensive equipment, reagents and trained professionals, most PCR assays are difficult to be implemented for on-site diagnosis. In this study, we developed a rapid and easy assay for KHV on-site diagnosis based on insulated isothermal PCR (iiPCR). KHV-specific primers and TaqMan probe were designed, targeting a highly conserved major capsid protein (MCP) region in KHV genome. KHVCPf1/r1 primers at 1:1 ratio resulted in the best sensitivity for KHV iiPCR. 100% (11/11) positive reactions were found in samples containing 102 copies of target DNA. The 67 bp iiPCR product was confirmed by DNA sequencing. The detection limit of the optimized KHV iiPCR was similar to real-time PCR using serial dilutions of two KHV positive samples. The specificity signals of KHV iiPCR were produced only from KHV DNA, but not CyHV1 and CyHV2. No significant interference from the fish genome DNA was detected. Validation of KHV iiPCR was done by analyzing 63 specimens side-by-side by iiPCR and real-time PCR. The results showed that 49 cases were identified as KHV-positive and 14 as KHV-negative samples by both assays. KHV iiPCR assay could be used as a simple, quick and inexpensive on-site diagnostic tool for KHV, making timely detection of KHV infection at the farm possible.
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