Establishment and application of TaqMan real-time fluorescence quantitative PCR for detecting the hybrid snakehead rhabdovirus

The purpose of this study is to establish a TaqMan real-time fluorescence quantitative PCR method which can be used to detect hybrid snakehead rhabdovirus( HSHRV). A coding region of HSHRVC1207 strain G protein was amplified by PCR and cloned into pMD18-T vector for the construction of recombinant plasmid. After being identified and confirmed by sequencing,the recombinant plasmids were extracted and 10-fold serial dilutions of recombinant plasmid were used as standard plasmid. A pair of specific primers and TaqMan probes were designed targeting the C1207 strain G gene. The standard plasmid was used as a quantitative template to establish the TaqMan real-time fluorescence quantitative PCR method for HSHRV detection and the sensitivity,repeatability and specificity of the method were evaluated. The results show ed that the TaqMan real-time fluorescence quantitative PCR method was established and the correlation coefficient( R2) and the slope of the standard curve were 0. 999 and-3. 290 respectively,which show ed a good linear relationship. Sensitivity tests show ed that the detection limit was 10 copies,w hich indicated that the sensitivity of the real time PCR is about 100 times higher than that of the conventional PCR assay. Results of repeatability tests show ed that the coefficient of variation was 0. 84%,indicating that this method had high repeatability. Furthermore,the method had high specificity for HSHRV without cross reactions with templates from other aquatic viruses. In clinical samples tests,detection rate of established fluorescence PCR was higher than that of conventional PCR. The TaqMan real-time fluorescence quantitative PCR established in this study has high sensitivity,repeatability and specificity for the rapid detection and quantification of HSHRV in fish.