摘要
建立适于基层实验室快速检测新型鸭呼肠孤病毒(Novel duck reovirus,NDRV)的一步反转录环介导等温扩增(RT-LAMP)方法。基于新型鸭呼肠孤病毒S3基因的6个保守区域设计了4条LAMP引物,利用Bst DNA聚合酶在63℃恒温保持45 min即可完成反转录和扩增反应,由此建立了RT-LAMP检测方法。该方法具有良好的特异性,除NDRV外对其他6种常见鸭病的检测结果均为阴性。该方法对病毒RNA的最低检出量为0.1 pg,是常规RT-PCR方法的100倍。临床应用结果表明,该方法与病毒分离鉴定方法的符合率为98%,而且对仪器的要求低,适于基层实验室和现场检测。
A one-step reverse transcription loop-mediated isothermal amplification （RT-LAMP） assay for detecting novel duck reovirus （NDRV） was established with 4 primers based on 6 conserved positions of the S3 gene. The process of assay was completed by using Bst DNA within 45 min at constant 63℃. RT-LAMP assay had solid specificity because no amplification was found with the samples of 6 other common duck diseases. The minimum detection limit of the RT-LAMP assay was 0.1 pg of viral RNA, which was 100 times of RT-PCR. The results of clinical application showed that the coincidence rate between the assay and the method of virus isolation and identification was 98%, and the requirement of instrument for the assay was relatively low. Therefore, the assay is a potential useful technique for NDRV detection in the field.
出处
《生物技术通报》
CAS
CSCD
北大核心
2015年第8期71-75,共5页
Biotechnology Bulletin
基金
山东省海外高层次人才(泰山学者)引进计划
山东省现代农业产业技术体系家禽创新团队计划(SDAIT-13-011-01)
公益性行业(农业)科研专项(201003012)
关键词
新型鸭呼肠孤病毒
S3基因
一步反转录环介导等温扩增
novel duck reovirus
S3 gene
one-step reverse transcription loop-mediated isothermal amplification
low requirement for instrument
