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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。
In order to establish a real-time TaqMan quantitative real-time PCR method that can detect HSHRV rapidly and accurately in clinic, the full-length sequence of HSHRV-C1207 G protein was amplified by PCR and the recombinant plasmid was constructed as fluorescence quantitative PCR was designed according to the conserved sequence of HSHRV-C1207 G protein. A pair of primer designed to amplify a 143 bp fragment and a TaqMan probe were designed and synthesized. The TaqMan real-time PCR assay for HSHRV was established using the standard as a template , And the sensitivity, repeatability and specificity of this method were evaluated. The results showed that the established standard curve of fluorescence quantitative PCR had a good linearity with a correlation coefficient (R2) of 0.999 and a slope of -3.290. A minimum of 10 copies of the viral nucleic acid molecule could be detected by fluorescence quantitative PCR, while the conventional PCR method The lowest detectable 1 × 103 copies; the coefficient of variation of 38 replicates in the replicate experiment group was 0.84%; there was no amplification reaction on the other 6 common aquaculture viruses. Twenty-one samples of the diseased samples were tested by this method, of which 18 were positive, similar to the results of cell separation and electron microscopy. Only 13 samples were positive by conventional PCR. The TaqMan real-time fluorescence quantitative PCR method established in this study is highly sensitive and specific and can be used to detect HSHRV clinically. It is of great significance for the quantitative detection of viral pathogens and the rapid diagnosis of hybrid Rana virus disease.