对虾传染性皮下及造血组织坏死病毒实时荧光LAMP检测方法的建立及应用  

Rapid Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus(IHHNV) by Real-time Fluorescence Loop-mediated Isothermal Amplification Assay

作　　者：李红梅[1,2] 江晓[1] 任春华[1] 赵哲[1] 胡超群[1] LI Hongmei JIANG Xiao REN Chunhua ZHAO Zhe HU Chaoqun(Key Laboratory of Tropical Marine Bio-resources and Ecology/ Key Laboratory of Applied Marine Biology of Guangdong Province, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, PR China University of Chinese Academy of Science Beijing 10004)

机构地区：[1]中国科学院南海海洋研究所/热带海洋生物资源与生态重点实验室/广东省应用海洋生物学重点实验室,广州510301 [2]中国科学院大学,北京100049

出　　处：《热带生物学报》2016年第3期307-313,共7页Journal of Tropical Biology

基　　金：广东省海洋经济创新发展区域示范专项项目(GD2012-B03-003);广东省省级科技计划项目(2014B030301064);"海洋微生物功能物质及其开发利用的研究"项目资助(KSCX2-EW-G-12B)

摘　　要：笔者研究构建了一种基于ESE-Quant tube scanner设备的新型实时荧光环介导等温扩增(loop-mediated isothermal amplification,LAMP)IHHNV检测方法,评价其敏感性、特异性、反应速度和实际检测结果。结果表明,IHHNV实时LAMP检测方法在63℃条件下,30 min内检测到106稀释的基因组DNA,可检测到24个质粒拷贝;与27种包括多种水生动物以及对虾常见病原DNA均无交叉反应。通过对152份实际样品的检测,实时荧光LAMP检测方法的检出阳性率为59.87%,与real-time PCR方法相当,高于其他检测方法。本研究建立的IHHNV实时LAMP检测方法对现场诊断具有较大的应用潜力。Infectious hypodermal and hematopoietic necrosis virus（ IHHNV） causes large economic losses to the shrimp aquaculture industry in the world. It is necessary to develop a fast,easy and accurate method to detect IHHNV. In this study,we developed a real-time fluorescence loop-mediated isothermal amplification assay for IHHNV detection on a small ESE-Quant Tube Scanner device and evaluated its sensitivity,specificity and rapidity. This method had a detection limit of 106 dilution template of genomic DNA within 30 min under isothermal condition at 63 C,and 24 copies of plasmid were detected. Furthermore,the approach had no non-specific signal response to other DNAs,including those of 27 species of aquatic animals and penaeid shrimp pathogens. This method had a positive detection rate of 59. 87%,similar to that of the real-time PCR but higher than other detection methods,and hence has great application potential in on-the-spot detection of IHHNV in shrimps.

关 键 词：传染性皮下及造血组织坏死病毒 实时荧光LAMP ESE-Quant TUBE SCANNER 

分 类 号：Q786[生物学—分子生物学] S917.4[农业科学—水产科学]