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罗氏沼虾野田村病毒(Macrobrachium rosenbergii nodavirus,MrNV)和双顺反子病毒(M.rosenbergii dicistrovirus,MrDV)是已报道对罗氏沼虾易感的主要致病性病毒,该研究通过建立双重RT-PCR方法对MrDV和MrNV两种病毒同时进行检测。根据MrDV和MrNV基因组序列的保守区分别设计特异性引物,并对双重PCR的退火温度和引物浓度进行优化,在获得优化反应体系和反应条件后,对罗氏沼虾样品进行检测。结果表明,双重PCR最佳退火温度为60℃,反应体系最佳引物终浓度MrNV384为0.1μmol/L,MrDV472为0.05μmol/L,对病样总RNA的最低检测限为360fg。引物的特异性检测表明,该检测方法对TSV、WSSV、IHHNV和嗜水气单胞菌TPS-30基因组无交叉反应。对阳性样品的病毒扩增序列分析表明,MrDV RNA依赖性RNA聚合酶编码区序列无变异,MrNV-RNA2序列存在较多变异,进化树结果表明2011年长三角的MrNV病毒主要来自于中国基因型和东南亚基因型。该方法的建立为罗氏沼虾病毒性疾病的预防和种苗的繁育奠定了基础。
Macrobrachium rosenbergii nodavirus (MrNV) and M.rosenbergii dicistrovirus (MrDV) are the major pathogenic viruses that have been reported to be susceptible to Macrobrachium rosenbergii. By establishing a dual RT- PCR method for MrDV and MrNV two viruses simultaneously detected. According to the conserved regions of MrDV and MrNV genome sequences, specific primers were designed respectively, and the annealing temperature and primer concentration of double PCR were optimized. After optimization reaction system and reaction conditions were obtained, samples of Macrobrachium rosenbergii were detected. The results showed that the optimum annealing temperature of dual PCR was 60 ℃. The optimal final concentration of MrNV384 was 0.1 μmol / L and MrDV472 was 0.05 μmol / L. The lowest detection limit of total RNA was 360 fg. The specificity of the primers showed that this method did not cross-react with TSV, WSSV, IHHNV and Aeromonas hydrophila TPS-30 genome. Sequence analysis of the positive samples showed that MrDV RNA-dependent RNA polymerase had no mutation in the coding region and MrNV-RNA2 sequence had many variations. The phylogenetic tree indicated that the MrNV virus in the Yangtze River Delta mainly came from the Chinese genotype in 2011 And Southeast Asian genotypes. The establishment of this method laid the foundation for the prevention of M. rosenbergii virus diseases and the breeding of seedlings.