虾肝肠胞虫LAMP检测技术的建立与应用  

Establishment and application of LAMP detection system for Enterocytozoon hepatopenaei

作　　者：陈进会 陈珍容 刘国庆 陈洵 段丽萍 谢会 石磊[3] CHEN Jin-hui;CHEN Zhen-rong;LIU Guo-qing;CHEN Xun;DUAN Li-ping;XIE Hui;SHI Lei(Huangpu Customs Technicul Center,Dongguan 523072,China;Animal Husbandry Bureau Zhongjiang Sichuan Province,Deyang 618101,China;Institute of Food Safety and Nutrition Ji Nan University,Guangzhou 510025,China)

机构地区：[1]黄埔海关技术中心,广东东莞523072 [2]四川省中江县畜牧局,四川德阳618107 [3]暨南大学食品安全与营养研究院,广东广州510025

出　　处：《中国兽医杂志》2019年第7期26-29,33,I0002,共6页Chinese Journal of Veterinary Medicine

基　　金：广东检验检疫局项目(2017GDK04)

摘　　要：本试验根据虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)18S的保守序列设计4套环介导等温扩增(LAMP)引物,从中筛选出一套引物对LAMP反应体系(Bst DNA聚合酶浓度、内外引物浓度比、dNTP浓度、Mg2+浓度)和反应条件(温度)进行优化。并对优化后的LAMP反应体系和条件进行特异性、灵敏度及精密度验证。特异性试验表明,该方法能特异性检测虾肝肠胞虫,而与其他病害的核酸没有交叉反应;灵敏度试验表明,可检测浓度为10 fg/μL的质粒DNA,制作的标准曲线可对虾肝肠胞虫进行定量分析。重复性试验结果表明,该方法Ct值的变异系数为2.88%~3.02%,具有良好的稳定性。而对于人工侵染的虾肝肠胞虫样品,LAMP方法可检测低至0.44 pg/μL。应用建立的LAMP方法对3个不同地区的120份凡纳滨对虾样品进行检测,检测结果表明,EHP阳性样品数为43,与qPCR检测结果符合率达97%。本试验所建立的LAMP方法具有特异性好、灵敏度高、重复性好的特点,并能在30 min内完成样品的检测,是快速、定量检测虾肝肠胞虫的有效手段。To screen the optimized primers for LAMP assay,a total of 4 sets specific primers were designed according to the conserved region sequence of Enterocytozoon hepatopenaei(EHP)18S gene,and the primer set EHP-1 was selected for further analysis.The optimal concentrations of Bst DNA polymerase,inner/outer primer ratio,dNTP and Mg2+of the LAMP reaction system and temperature of the LAMP reaction condition were screened by combining a single factor experiment.The specificity of this LAMP assay was confirmed by negative testing for other nucleic acid.The detection sensitivity of the EHP by the species-specific primer is 10 fg/μL.The standard curve obtained from the reaction exhibited a good linearity.Repeatability tests showed that threshold cycle of the method gave a good stability with the coefficient of the variations between assays being within 2.88%to 3.02%.In addition,the established method was used for the detection of 120 samples,and 43 samples were EHP positive,which were 97%coincidence rate with RT-PCR.The proposed LAMP method developed in this study could be more convenient for use in the field as a simple,rapid,and effective technique for monitoring Enterocytozoon hepatopenaei within 30 min.

关 键 词：虾肝肠胞虫 环介导等温扩增(LAMP) 18S qPCR 

分 类 号：S852.723[农业科学—基础兽医学]