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基因Ⅱ型草鱼呼肠孤病毒(GCRV-Ⅱ)是当前引起草鱼出血病暴发与流行的主要病原,为建立快速检测GCRV-Ⅱ的方法,本研究根据GCRV-ⅡRd Rp基因的保守区域序列设计引物及TaqMan探针,经反应参数优化,建立了检测GCRV-Ⅱ的荧光定量PCR方法。结果显示,该方法仅对GCRV-Ⅱ的靶基因序列进行扩增,与其它非靶目标核酸均无交叉反应;其最低检出量为3拷贝/μL病毒核酸,比普通PCR的敏感度高100倍;组内和组间重复试验变异系数均小于1%。采用该方法检测60份草鱼出血病疑似样品,GCRV-Ⅱ阳性检出率为75%(45/60),与细胞分离结果一致。应用该方法分析了GCRV-Ⅱ在不同细胞和不同培养方式下病毒的增殖含量,结果显示GSB和PSF细胞增殖量最大,达106拷贝/μL~107拷贝/μL病毒核酸,转瓶接种比常规的细胞培养瓶和培养板接种其病毒含量低2个数量级。本研究建立的GCRV-ⅡTaqMan荧光定量PCR方法具有高效、特异、敏感、可重复性强的优点,适用于GCRV-Ⅱ的临床快速检测和病毒定量分析。
In order to establish a rapid method for the detection of GCRV-Ⅱ, the GCRV-Ⅱ gene is the main pathogen causing the outbreak and epidemic of grass carp hemorrhagic disease. In this study, primers were designed based on the conserved region of GCRV-ⅡRd Rp gene And TaqMan probe. After the reaction parameters were optimized, a fluorescence quantitative PCR method for detecting GCRV-Ⅱ was established. The results showed that this method only amplified the target gene sequence of GCRV-Ⅱ and did not cross-react with other non-target nucleic acids. The detection limit was 3 copies / μL of viral nucleic acid, which was 100 Times; the coefficient of variation of repeated tests in both groups were less than 1%. Using this method to detect 60 suspected grass carp hemorrhage samples, the positive detection rate of GCRV-Ⅱ was 75% (45/60), which was consistent with the result of cell separation. Using this method, the proliferation of GCRV-Ⅱ in different cells and different culture modes was analyzed. The results showed that the proliferation of GSB and PSF cells was the highest, reaching 106 copies / μL to 107 copies / μL of viral nucleic acid. Cell culture flasks and plates inoculated with its virus content two orders of magnitude lower. The established GCRV-ⅡTaqMan fluorescence quantitative PCR method has the advantages of high efficiency, specificity, sensitivity and repeatability, and is suitable for the rapid clinical detection of GCRV-Ⅱ and the quantitative analysis of the virus.