可视化的RT-LAMP方法检测禽白血病病毒  

Visualized Detection of Avian Leukosis Virus by Reverse Transcription Loop-mediated Isothermal Amplification Assay

作　　者：刘业兵[1] 张磊[1] 孙跃辉[2] 毛娅卿[1] 李俊平[1] 宁宜宝[1] 

机构地区：[1]中国兽医药品监察所,北京100081 [2]天津大学,天津300072

出　　处：《畜牧兽医学报》2011年第1期150-156,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：2010年公益性行业(农业)科研专项(201003010);十一五国家科技支撑计划(2007BAD86B04-4)

摘　　要：本研究旨在建立一种快速检测禽白血病病毒(ALV)的RT-LAMP方法。根据GenBank中ALV序列,在其保守序列区域设计了多套LAMP引物,通过LAMP Real-time Turbidimeter仪对ALV的LAMP引物、反应体系及反应进程进行监测和分析,以评价该方法的特异性和敏感性,并对加入SYBR GreenⅠ肉眼判定与仪器监测结果进行比对。结果表明:建立的RT-LAMP方法在63℃水浴中36min可对ALV核酸进行高效扩增,在反应结束后加入SYBR GreenⅠ肉眼判断结果与Real-time Turbidimeter仪检测结果一致;该方法具有很强特异性,对其它相关鸡病病原检测结果均为阴性;其检出限量为14.2pg.μL-1,显示出很高的敏感性;用建立的LAMP方法对24个样品进行检测,结果表明LAMP法与其原检测结果基本一致;本研究建立了可对ALV进行快速检测并可肉眼判定结果的可视化RT-LAMP方法,其操作简便、无需昂贵设备,适用于ALV的快速检测。We established a rapid detection of avian leukosis virus（ALV） by reverse transcription-loop-mediated isothermal amplification assay（RT-LAMP） in this study.According to the published ALV sequences in GenBank,multiple pairs of primers were designed targeting the conserved region of ALV.The primer,reaction system and processes of LAMP were detected and analyzed by the LAMP Real-time Turbidimeter so as to evaluate its specificity and sensitiveness.Meanwhile,the amplified products were colored by SYBR GreenⅠ after completion of the reaction,so that the amplification can be detected with naked eyes and the results were compared to the former detected by LAMP Real-time Turbidimeter.RT-LAMP showed a highly efficient amplification for ALV nucleic acid which performed at 63 ℃ for 36 min.The results detected with naked eyes by the addition of SYBR GreenⅠ at the end of reaction were consistent with the results detected by Turbidimeter.The RT-LAMP assay showed a strong specificity that the results were all negative towards other nosazontology detection in chicken;and it also showed a high sensitivity with a detection limit of 14.2 pg·μL-1.The results of 24 clinical samples detected by LAMP developed in this study were consistent with the ones tested before by ELISA and COFAL test.A visualized RT-LAMP method which can detect ALV rapidly was established and the results can also be read with naked eyes.The method was easy to operation,which was suitable for rapid detection of ALV.

关 键 词：禽白血病病毒 环介导等温扩增技术(LAMP) 检测 

分 类 号：S852.659.3[农业科学—基础兽医学] S858.315.3[农业科学—兽医学]