杂交鳢(斑鳢♀×乌鳢♂)弹状病毒TaqMan实时荧光定量PCR检测方法的建立及应用

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

为了建立一种能在临床上快速、准确检测杂交鳢弹状病毒(HSHRV)的TaqMan实时荧光定量PCR方法,利用PCR技术扩增HSHRV-C1207 G蛋白的全长序列,构建重组质粒,作为荧光定量PCR的标准品,根据HSHRV-C1207 G蛋白的保守序列,设计合成了一对能特异性扩增143 bp片段的引物和TaqMan探针,以标准品为模板建立HSHRV的TaqMan实时荧光定量PCR检测方法,并对该方法的灵敏度、可重复性和特异性进行评价。结果显示:建立的荧光定量PCR检测方法标准曲线有较好的线性关系,相关系数(R2)为0.999,斜率为-3.290;荧光定量PCR最低可以检测到10个病毒核酸分子拷贝,而传统PCR方法最低可检测到1×103个拷贝;38个平行样品重复性实验组内变异系数为0.84%;对其他6种水产养殖常见病毒均无扩增反应。应用该方法对采集的21份患病鳢样品进行检测,其中18份为阳性,与细胞分离和电镜观察结果相同;以传统PCR方法检测同样的样品,仅13份为阳性。本研究建立的TaqMan实时荧光定量PCR方法灵敏度高、特异性强,能较好的用于临床HSHRV的检测,对病毒病原定量检测与杂交鳢弹状病毒病的快速诊断具有重要意义。

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 HSHRV- C1207 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 showed 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 showed a good linear relationship. Sensitivity tests showed that the detection limit was 10 copies, which 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 showed 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.