【摘要】 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。更多还原

【Abstract】 Macrobrachium rosenbergii nodavirus(MrNV) and Macrobrachium rosenbergii dicistrovirus(MrDV) are the main viral pathogens of Macrobrachium rosenbergii.In order to detect the two viruses,two pairs of primers targeted sequences located within the MrNV capsid protein gene and MrDV RNA-dependent RNA polymerase(RDRP) gene amplified amplicons of 384 bp and 472 bp,respectively.The reaction conditions of the duplex RT-PCR were optimized and PCR products were sequenced.Meanwhile,the specificity and sensitivity of duplex RT-PCR were studied.Results reveal that optimum annealing temperature of duplex RT-PCR is 60 ℃ and the minimum concentrations of primers are 0.1 μmol/L of MrNV384 and 0.05 μmol/L of MrDV472.The detection limit of duplex RT-PCR was determined to be 360 fg of tissue total RNA for both the viruses.The two pairs of primers were found to be specific to MrNV and MrDV respectively,and did not to react to either Taura syndrome virus(TSV),White spot syndrome virus(WSSV),Infectious hypodermal and hematopoietic necrosis virus(IHHNV),or Aeromonas hydrophila TPS-30.Sequence analysis of RDRP gene amplicons of MrDV revealed no variations.By contrast,sequence analysis of MrNV amplicons showed many variations in nucleotide sequences of the capsid protein gene.The phylogenetic tree shows that the stains of MrNV,collected from Yangtze River delta in 2011,are from two genotypes,Chinese and Southeast Asia genotypes.This new duplex RT-PCR method could be applied for routine health monitoring,early virus detection,and studying virus-host interaction,detection of carriers and screening of broodstock.更多还原