Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid and real-time detection of Japanese encephalitis virus
Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid and real-time detection of Japanese encephalitis virus
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DOI：
10.1128/jcm.01487-06
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发表时间：
2006-11-01
影响因子：
9.4
通讯作者：
Morita, Kouichi
中科院分区：
文献类型：
--
作者：
Parida, M. M.;Santhosh, S. R.;Morita, Kouichi
据报道，一步、单管加速定量逆转录环介导等温扩增 (RT-LAMP) 测定的标准化和验证可用于快速实时检测日本脑炎病毒 (JEV)。本研究报告的 RT-LAMP 检测非常简单且快速；使用针对JEV E基因的一组6个引物，在63℃等温条件下，可以在30分钟内获得扩增。与 RT-PCR 相比，RT-LAMP 检测显示出极高的灵敏度，检测限为 0.1 PFU。所选引物组的特异性是通过与 JEV 血清复合物其他密切相关成员的交叉反应性研究以及对健康人类志愿者的评估来确定的。使用有限数量的患者脑脊液样本对 RT-LAMP 检测进行临床诊断的比较评估显示，与传统 RT-PCR 的一致性为 85%，灵敏度和特异性分别为 100% 和 86%。根据样品中阳性时间的标准曲线确定，大多数临床样品中的病毒浓度为 10(2) 至 10(5) PFU/ml。此外，使用SYBR green I荧光染料也可以用肉眼可视化对基因扩增的监测。因此，由于操作简单，不需要复杂的设备和技术人员，本文报道的 RT-LAMP 检测是一种快速、实时检测 JEV 的宝贵工具，不仅适用于设备精良的实验室，而且适用于发展中国家财力有限的外围诊断实验室。
The standardization and validation of a one-step, single-tube accelerated quantitative reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay is reported for rapid and real-time detection of Japanese encephalitis virus (JEV). The RT-LAMP assay reported in this study is very simple and rapid; the amplification can be obtained in 30 min under isothermal conditions at 63 degrees C by employing a set of six primers targeting the E gene of JEV. The RT-LAMP assay demonstrated exceptionally higher sensitivity compared to that of RT-PCR, with a detection limit of 0.1 PFU. The specificities of the selected primer sets were established by cross -reactivity studies with other closely related members of the JEV serocomplex as well as by evaluation of healthy human volunteers. The comparative evaluation of the RT-LAMP assay for clinical diagnosis with a limited number of patient cerebrospinal fluid samples revealed 85% concordance with conventional RT-PCR, with a sensitivity and a specificity of 100% and 86%, respectively. The concentration of virus in most of the lclinical samples was 10(2) to 10(5) PFU/ml, as determined from the standard curve based on the time of positivity in the samples. In addition, the monitoring of gene amplification can also be visualized with the naked eye by using SYBR green I fluorescent dye. Thus, due to easy operation without a requirement of sophisticated equipment and skilled personnel, the RT-LAMP assay reported here is a valuable tool for the rapid and real-time detection of JEV not only by well-equipped laboratories but also by peripheral diagnostic laboratories with limited financial resources in developing countries.