WHO Japanese Encephalitis Regional Reference Laboratory for the Western Pacific Region/Division of Arboviruses, National Institute of Health, Korea Centers for Disease Control and Prevention, Seoul, Republic of Korea.
J Virol Methods. 2011 Jan;171(1):248-52. doi: 10.1016/j.jviromet.2010.11.009. Epub 2010 Nov 17.
A novel nested reverse transcription-polymerase chain reaction (RT-PCR)-based kit is described for detecting Japanese encephalitis virus (JEV), especially for genotype 1 and 3 strains. The assay consists of a first round RT-PCR and a subsequent nested PCR amplification. It has unique features such as the use of a premix system in which all reagents are lyophilized in reaction tubes and the inclusion of control RNA in each reaction to monitor false negative results. In addition, an automatic tissue homogenizer and a RNA extraction system are used concurrently for assay standardization and increasing throughput. The assay using the kit proved specific for JEV with no amplification of other JEV-related flaviviruses. The detection limits were approximately 0.1 PFU/ml and 1 PFU/ml for JEV genotypes 1 and 3, respectively. The assay protocol has been validated in large-scale field trials in South Korea during the 2008-2009 surveillance seasons. Nineteen of 1136 pools of mosquitoes (54,583 mosquitoes total) were identified as JEV positive. This nested RT-PCR kit combined with control RNA and an automatic RNA extraction system should be suitable for routine JEV surveillance programs.
一种新型的巢式逆转录-聚合酶链反应（RT-PCR）试剂盒被用于检测日本脑炎病毒（JEV），尤其是针对基因型 1 和 3 株。该检测方法由第一轮 RT-PCR 和随后的巢式 PCR 扩增组成。它具有独特的特点，如使用预混系统，其中所有试剂都在反应管中冻干，以及在每个反应中包含对照 RNA 以监测假阴性结果。此外，还同时使用自动组织匀浆器和 RNA 提取系统来进行检测标准化和提高通量。该试剂盒的检测方法证明对 JEV 具有特异性，不会扩增其他 JEV 相关黄病毒。对于 JEV 基因型 1 和 3，检测限分别约为 0.1 PFU/ml 和 1 PFU/ml。该检测方案已在韩国 2008-2009 年监测季节的大规模现场试验中得到验证。在总共 54583 只蚊子的 1136 个蚊虫池中，有 19 个被鉴定为 JEV 阳性。这种巢式 RT-PCR 试剂盒结合对照 RNA 和自动 RNA 提取系统，应该适用于常规的 JEV 监测计划。
