仙台病毒RT-LAMP可视化检测方法的建立  

Establishment of a visualized detection method of Sendai virus by reverse transcription loop-mediated isothermal amplification

作　　者：周洁 赵丽娟[1,2] 陶凌云 倪丽菊 高诚 陈洪岩[3] 

机构地区：[1]上海实验动物研究中心,上海201203 [2]扬州大学兽医学院,扬州225009 [3]中国农业科学院哈尔滨兽医研究所实验动物中心,哈尔滨150001

出　　处：《中国实验动物学报》2016年第3期293-298,共6页Acta Laboratorium Animalis Scientia Sinica

基　　金：上海市科委科技创新行动计划(11140901000);上海市科委研发平台专项(15DZ2292400)

摘　　要：目的建立一种快捷、灵敏的检测方法,即反转录-环介导等温扩增方(RT-LAMP)用于仙台病毒(SeV)的检测。方法根据Gen Bank公布的SeV序列(DQ219803.1),在其保守区域设计了六套LAMP引物,利用LAMP Real-Time Turbidimeter LA-320C仪监测反应进程并筛选最佳引物、反应条件,建立对SeV核酸进行特异性扩增的RT-LAMP检测方法,并可通过加入目测荧光检测试剂肉眼判断结果。对所建立的方法进行了敏感性、特异性评估并对92份样品进行了检测。结果该方法在63℃恒温下作用60 min,SeV RNA获得了高效率的特异性扩增,与其余常见小鼠病毒无交叉反应;最低检出量为2.1TCID_(50) SeV,比RT-PCR方法高102倍;肉眼判断结果与RealTime Turbidimeter LA-320仪监测结果一致;通过对92份样品的RT-LAMP,RT-PCR和间接ELISA方法的检测比对,三者符合率为100%。结论建立的SV RT-LAMP检测方法具有快速、特异、灵敏,操作简单的特点,具有良好的应用前景。Objective To establish a simple and sensitive detection method of Sendai virus（ SeV） by reverse transcription loop-mediated isothermal amplification（ RT-LAMP） technique. Methods According to the published GenBank sequences（ DQ219803. 1）,six pairs of primers were designed targeting the conserved region of SeV. The amplification products were detected with a LAMP real-time Turbidimeter.（ LA-302）. Through optimizing the LAMP primers and reaction conditions,a rapid and specific detection method of SeV was established. Meanwhile,the amplified products were colored by fluorescence detection reagent after completion of the reaction,so that the amplification could be visualized and detected by naked eyes. Then,methodological evaluation of the RT-LAMP was tested. Results The method of RT-LAMP showed a highly efficient amplification for SeV viral target gene which was performed at 63℃ for 60 min with the LAMP real-time Turbidimeter（ LA-302）. The detection limit was 2. 1 TCID50,100 times higher than that of RT-PCR,and no crossreaction with other RNA and DNA viruses of mice was observed. The results of SeV LAMP reaction was visualized and the tube could be directly observed by naked eyes with the addition of fluorescence detection reagent. The results were consistent with the results detected by real-time tubidimeter. 92 clinical samples were detected byRT- LAMP,RT-PCR and indirect ELISA,and the coincidence rate was 100%. Conclusions This established SeV RT-LAMP detection method is fast,specific,highly sensitive,easy to perform under simple conditions,and is suitable for rapid detection of Sendai viirus.

关 键 词：仙台病毒 RT-LAMP 实时监控 快速检测 

分 类 号：Q95-33[生物学—动物学]