实时荧光LAMP法检测转基因猪的标记基因hCD3  

Real-time Fluorescence Loop-mediated Isothermal Amplification for Detection of hCD39 in Transgenic Pig

作　　者：李津[1] 刘巍[1] 李俊超[2] 金红岩[1] 李刚[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所农业部兽用药物与兽医生物技术科学观测实验站,北京100193 [2]中国人民解放军总医院心血管内科,北京100853

出　　处：《畜牧兽医学报》2017年第3期538-543,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家转基因重大专项(2014ZX0801203B-002);国家自然科学基金(31472203);国家科技支撑计划(2013BAD12B05);FAO/IAEA国际合作项目(17453/D32025)

摘　　要：利用ESE-Quant tube scanner监测平台,建立了一种基于实时荧光LAMP技术的hCD39基因快速检测方法。针对hCD39基因设计6条特异性引物,在63℃条件下,进行核酸扩增反应45 min。在扩增前加入SYBR GreenⅠ染料作为反应的指示剂,以SYBR GreenⅠ染料的荧光强度作为结果判定标准。该方法灵敏度高,最小可检测到3fg·μL-1;特异性好,与转基因猪中的标记基因hCD46、LEA29Y均无交叉反应;并且可对口腔拭子标本进行检测。因此,研究建立的实时荧光LAMP法,操作简单,反应快速,灵敏度高,特异性好,并且可以对猪活体进行无损伤采样检测,适合在现场快速简便的操作。Using ESE-Quant tube scanner testing platform,we established a real-time fluorescence loop-mediated isothermal amplification（RTF-LAMP）method for rapid detection of hCD39.The method employed a set of six specially designed primers for amplification of nucleic acid under isothermal conditions at 63℃for 45 min.The amplification process of LAMP was monitored by the addition of SYBR GreenⅠ dye prior to amplification.The fluorescence intensity of SYBR GreenⅠ dye was used as the standard of the results.Results of the study showed that the sensitivity of the method was 3fg·μL-1,and there was no cross-reactivity with the genes of related and unrelated detected,such as hCD46,LEA29 Y.More importantly,this method could be completed testing of buccal swabs specimens of transgenic pigs.The purpose of our study was to develop a simple,economical,rapid method with good sensitivity and specificity.The method could be no damage to the living body samples for testing,suitable for quick and easy on-site operation.

关 键 词：环介导等温扩增(LAMP) 特异性检测 hCD39 

分 类 号：S858.91[农业科学—临床兽医学]