作　　者： ; ; ; ; ; ; ; ; ;

机构地区： 深圳出入境检验检疫局

出　　处： 《中国水产科学》 2008年第3期506-510,共5页

摘　　要： 根据GenBank中登录的鱼类神经坏死病毒CP基因序列,选择高度保守区域设计引物和TaqMan荧光探针,通过对实时荧光RT-PCR反应条件进行优化,建立了用于检测鱼类神经坏死病毒的实时荧光RT-PCR方法。利用该方法检测鱼类神经坏死病毒及其他多种常见的水生动物RNA病毒,结果只能检测到目的病毒,表明其具有良好的特异性。灵敏性试验发现,其最低检测限可达1.2pg/μL的总RNA。与RT-PCR的灵敏度对比试验表明,其敏感度比RT-PCR高100倍。对同一样品进行检测,在组内及组间的变异系数分别为0.9%以及1.5%,证实其重复性极好,并且从抽提核酸到得出结果仅需4h。对临床500份样品进行鱼类神经坏死病毒检测,结果发现有40份阳性样品。这些结果表明,本研究所建立的实时荧光RT-PCR能对鱼类神经坏死病毒进行准确、快速的检测,具有特异性好、灵敏度高的优点,是开展鱼类神经坏死病的临床检测和疫情监测工作的有力工具。 Viral nervous necrosis （VNN） has already become an emerging problem in several fanned marine fish species in various geographic areas all over the world. The causative viral agents consisted of one major coat protein of approximately 42 kD and two single-strand positive-sense RNAs without poly （A） structure at the 3＇ -terminus. Recently, reverse transcription-polymerase chain reaction （RT-PCR） has been developed to detect viral nervous necrosis viruses, in which false positive may be resulted from contamination. In this study, a real-time RT-PCR assay for fish nervous necrosis virus was developed. The specific primers and TaqMan probes were designed according to the highly conservative sequence of capsid protein （CP） gene of viral nervous necrosis virus from GenBank. After the procedure of the real-time RT-PCR assay for fish nervous necrosis virus was optimized, the specificity, sensitivity, and reproducibility of the method was estimated. It was found that the specificity of this assay was high without any cross-reactions with SVC, IPN, VHSV, GCHV, IHNV, and so on. A minimum of 1.2 pg/pL total RNA could be detected by the assay, whose sensitivity was 100 times than that of RT-PCR. The coefficients of variance （CVs） were 0.9% and 1.5% for the intra-assay and inter-assay tests respectively, which indicated good reproducibility. It took only 4 h to complete the whole course of reaction including extraction of viral RNA and the real-time RT-PCR. Detected with real-time RT-PCR assay for fish nervous necrosis virus, it was found that forty specimen were positive among 500 clinical samples. Those results confirm that the real-time RT-PCR assay for VNNV is a rapid, sensitive and specific method for the detection of viral nervous necrosis virus. [Journal of Fishery Sciences of China, 2008, 15 （3） ： 506-510]

关 键 词： 鱼类神经坏死病毒 实时荧光 检测方法

领　　域： [农业科学] [农业科学]