新型鸭呼肠孤病毒RT-LAMP检测方法的建立

doi:10.13560/j.cnki.biotech.bull.1985.2015.08.011

生物技术通报 ›› 2015, Vol. 31 ›› Issue (8): 71-75.doi: 10.13560/j.cnki.biotech.bull.1985.2015.08.011

新型鸭呼肠孤病毒RT-LAMP检测方法的建立

于可响¹, 马秀丽¹, 韩宏宇^{1, 2}, 刘存霞¹, 李玉峰¹, 黄兵¹, 宋敏训¹

1.山东省农业科学院家禽研究所山东省家禽疫病诊断与免疫重点实验室,济南 250023; 2.山东农业大学动物科技学院,泰安 271018

收稿日期:2014-12-10 出版日期:2015-08-21 发布日期:2015-08-22
作者简介:于可响,男,博士,副研究员,研究方向：禽病诊断与免疫;E-mail：yukx1979@163.com
基金资助:
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）

A RT-LAMP Assay for Detection of Novel Duck Reovirus

Yu Kexiang¹, Ma Xiuli¹, Han Hongyu^{1, 2}, Liu Cunxia¹, Li Yufeng¹, Huang Bing¹, Song Minxun¹

1. Key Laboratory of Poultry Disease Diagnose and Immune of Shandong Province,Institute of Poultry,Shandong Academy of Agricultural Sciences,Ji’nan 250023; 2. College of Animal Science ＆Veterinary Medicine,Shandong Agricultural University,Taian 271018

Received:2014-12-10 Published:2015-08-21 Online:2015-08-22

摘要/Abstract

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。

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

Abstract: 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.

Key words: novel duck reovirus, S3 gene, one-step reverse transcription loop-mediated isothermal amplification, low requirement for instrument

于可响, 马秀丽, 韩宏宇, 刘存霞, 李玉峰, 黄兵, 宋敏训. 新型鸭呼肠孤病毒RT-LAMP检测方法的建立[J]. 生物技术通报, 2015, 31(8): 71-75.

Yu Kexiang, Ma Xiuli, Han Hongyu, Liu Cunxia, Li Yufeng, Huang Bing, Song Minxun. A RT-LAMP Assay for Detection of Novel Duck Reovirus[J]. Biotechnology Bulletin, 2015, 31(8): 71-75.

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新型鸭呼肠孤病毒RT-LAMP检测方法的建立

A RT-LAMP Assay for Detection of Novel Duck Reovirus

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