Development and field evaluation of a nested RT-PCR kit for detecting Japanese encephalitis virus in mosquitoes

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.