罗氏沼虾诺达病毒套式RT-PCR检测方法的建立及初步应用

Establishment and preliminary application of a nested RT-PCR for the detection of Macrobrachium rosenbergii nodavirus

罗氏沼虾诺达病毒(Macrobrachium rosenbergii nodavirus,MrNV)是世界动物卫生组织规定必需上报的罗氏沼虾白尾病(whitetail disease,WTD)的主要病原体。为建立快速检测MrNV的套式RT-PCR方法,根据GenBank上发表的MrNV衣壳蛋白基因序列设计2对特异性引物,对PCR条件进行优化后通过特异性试验和敏感性试验检测其特异性和敏感性。结果表明:该方法能特异性地扩增出205 bp的MrNV衣壳蛋白基因片段,最佳引物浓度为0.8μmol/L、退火温度为56℃、Mg2+浓度为2.0 mmol/L,而对其他对虾病毒基因组均没有扩增出条带;检测MrNV的灵敏度大约为RT-PCR的103倍,最低可检测到2.612×10-2 fg MrNV RNA。应用套式RT-PCR和一步法RT-PCR同时对108份来自广西各地的罗氏沼虾临床样品进行检测,其中套式RT-PCR共有9份检出MrNV,而一步法RT-PCR仅有3份检出MrNV。由此可见,建立的套式RT-PCR检测方法具有极高的特异性和敏感性,能提高MrNV的阳性检出率,可用于MrNV急性感染和隐性感染的早期诊断,为WTD的临床检测、流行病学调查、进出口检疫和实验室研究提供可靠的技术手段。研究亮点:根据GenBank上发表的MrNV衣壳蛋白基因序列设计2对特异性引物,优化建立了检测MrNV的套式RT-PCR方法。该方法具有极高的特异性和敏感性,最低可检测到2.612×10-2fg MrNV RNA,能提高临床样品MrNV的阳性检出率,可用于MrNV急性感染和隐性感染的早期诊断。

Macrobrachium rosenbergii nodavirus（MrNV） was the main pathogen of white tail disease（WTD）,which was defined as the notifiable disease of aquatic animals by Office International des Epizooties（OIE）.The purpose of this study is to develop a nested RT-PCR method for the detection of MrNV.Two pairs of specific primer were designed and synthesized according to the published MrNV capsid protein gene sequence in GenBank.After the nested RT-PCR method was optimized,the specificity and sensitivity were assayed.This method can produce 205 bp amplicons of MrNV capsid protein gene with the following conditions： 0.8 μmol/L optimal concentration of primer,56 ℃ anneal temperature and 2.0 mmol/L Mg2＋ concentration,and had no cross-reaction with three other prawn pathogens such as taura syndrome virus（TSV）,white spot syndrome virus（WSSV） and infectious hypodermal and haematopoietic necrosis virus（IHHNV）.The results of sensitivity test showed that the nested RT-PCR was 103 times more sensitive than that of one-step RT-PCR,and the minimum detection limits of the nested RT-PCR were 2.612×10-2fg MrNV RNA.108 clinical Macrobrachium rosenbergii samples from Guangxi were tested by the two RT-PCR,and 9 out of 108 samples were positive by nested RT-PCR,while 3 out of 108 samples by one-step RT-PCR,which showed that the nested RT-PCR can boost positive detection rate of MrNV.The results indicated that the nested RT-PCR method for the detection of MrNV was successfully established and this method has high specificity and sensitivity,and could be used for the early diagnosis of acute and latent infections of MrNV.This study provides a reliable way for clinical detection,epidemiological investigation,import and export quarantine and laboratory studies on MrNV.