罗氏沼虾野田村病毒中国株RT-LAMP-LFD快速检测方法的研究

Rapid Detection of Macrobrachium Rosenbergii Nodavirus Isolated in China by a Reverse-transcription Loop-mediated Isothermal Amplification Assay Combined with a Lateral Flow Dipstick Method

罗氏沼虾白尾病(White tail disease,WTD)是流行于罗氏沼虾苗种阶段的重要疾病,该病病原被确定为罗氏沼虾野田村病毒(Macrobrachium rosenbergii Nodavirus,MrNV),为了建立罗氏沼虾野田村病毒中国分离株(MrNV-chin)的便捷、灵敏、特异的分子诊断方法,本研究根据罗氏沼虾野田村病毒RNA2基因保守序列的6个区域设计了4条特异性引物和1条检测探针,将环介导等温扩增技术与侧向流层析试纸(Lateral flow dipstick,LFD)相结合,建立了MrNV的RT-LAMP-LFD检测方法。结果表明,该方法的灵敏度是常规琼脂糖凝胶电泳的10倍;从核酸提取到检测结果判定仅需45min;反应特异性强,对其他虾类病毒WSSV、IHHNV、IMNV、TSV和YHV扩增结果均为阴性;操作简单,不需要复杂的仪器,在61℃水浴反应即可;利用LFD试纸显示结果,肉眼可直接观察判定。作为一种快速灵敏实用的分子诊断技术,该方法有利于罗氏沼虾白尾病的诊断与预防。

White coloration of the muscle of the giant river prawn （Macrobrachium rosenbergii） is a serious problem in China. The Macrobrachium rosenbergii Nodavirus （MrNV） has been confirmed to be the pathogen that causes this disorder. To develop a rapid, sensitive and specific technology for the detection of Macrobrachium rosenbergii Nodavirus isolated from China （MrNV-China）, a reverse-transcription loop- mediated isothermal amplification assay combined with a lateral flow dipstick （RT-LAMP-LFD） assay method is described. A set of four primers and a labeled probe were designed specifically to recognize six distinct regions of the MrNV RNA2 gene. Results showed the sensitivity of the RT-LAMP-LFD assay was ten-times higher than the reverse-transcription loop-mediated isothermal amplification assay （RT-LAMP） with agarose gel electrophoresis. The assay was conducted with one-step amplification at 61℃ in a single tube within 45 min. No product was generated from shrimps infected with other viruses, including DNA viruses （infectious hypodermal and hematopoietic necrosis virus （IHHNV）; white spot syndrome virus （WSSV）） and RNA viruses （Taura syndrome virus （TSV） ; infectious myonecrosis virus （IMNV） ; yellow head virus （YHV））. Results were visualized by the LFD method. Therefore, the described rapid and sen- sitive assay is potentially useful for MrNV detection.