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草鱼呼肠孤病毒逆转录环介导等温扩增(RT-LAMP)检测方法的建立
作者:
作者单位:

张金凤1, 曾令兵1, 2, 张辉2, 周勇2, 肖艺2, 苏岚1, 高正勇12. 中国水产科学研究院 长江水产研究所, 湖北 武汉 430223

作者简介:

张金凤(1987−), 女, 硕士研究生, 主要从事水产养殖病害研究. E-mail: zhang-jin-feng@126.com

中图分类号:

S941

基金项目:

国家现代农业产业技术体系建设专项资金资助项目(CARS-46-11).


Development of a reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for detection of grass carp reovirus
Author:
Affiliation:

1. Huazhong Agricultural University, Wuhan 430070, China;2. Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Wuhan 430223, China

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    摘要:

    根据草鱼呼肠孤病毒, (RT-LAMP)1 h绿色阳性结果明显区别于橙色阴性结果。该检测体系针对草鱼呼肠孤病毒的检测灵敏度高, , 为快速检测草鱼呼肠孤病毒与诊断草鱼出血病提供了简捷快速的技术手段。

    Abstract:

    Two pairs of specific primers were designed based on the grass carp reovirus (GCRV) VP6 coding gene sequence. Using GCRV genomic total RNA as a template, we developed a reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay. After optimizing the reaction conditions, we were able to successfully amplify the targeted GCRV VP6 coding gene at 63 in 1 h. Analysis of the amplified products by agarose electrophoresis revealed that the band pattern resembled that of the ladder diagram in a gel. After adding SYBR Green I fluorescent dye to the reaction system, the green positive amplification could be viewed by eye. Our assay was highly sensitive to grass carp reovirus with a lower detection limit of 33 pg, which was 10-fold higher than that of traditional RT-PCR for GCRV. Moreover, the assay was specific for the detection of GCRV and was not susceptible to cross reaction with other viruses, including Channel catfish reovirus (CCRV), Spring viremia of carp virus (SVCV), Koi herpesvirus (KHV), and giant salamander iridovirus (GSIV). In conclusion, the RT-LAMP assay is convenient, rapid, sensitive, and specific for GCRV detection. Our assay provides a novel approach for the detection of GCRV and the diagnosis of grass carp hemorrhage.

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张金凤,曾令兵,张辉,周勇,肖艺,苏岚,高正勇.草鱼呼肠孤病毒逆转录环介导等温扩增(RT-LAMP)检测方法的建立[J].中国水产科学,2013,20(1):129-136
ZHANG Jinfeng, ZENG Lingbing, ZHANG Hui, ZHOU Yong, XIAO Yi, SU Lan, GAO Zhengyong. Development of a reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for detection of grass carp reovirus[J]. Journal of Fishery Sciences of China,2013,20(1):129-136

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  • 在线发布日期: 2013-02-07
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