| Abstract: | Japanese encephalitis (JE) is a disease that threatens both human and animal populations in Asian countries, and the causative agent of JE, Japanese encephalitis virus (JEV), has recently changed from genotype III (GIII) to genotype I (GI). However, a test for the rapid differentiation of GI and GIII JEV is still unavailable, especially one that can be used for mosquito‐based surveillance. We have designed GI‐ and GIII‐specific primer sets for the rapid detection and differentiation of GI and GIII JEV by multiplex reverse transcriptase‐polymerase chain reaction (multiplex RT‐PCR). The GI‐specific and GIII‐specific primer sets were able to specifically amplify the target gene from GI and GIII JEV, respectively. The limitations of detection were 0.00225 and 0.225 pfu for the GI‐specific and GIII‐specific primers, respectively. Using a mixture of GI‐specific and GIII‐specific primers, the multiplex RT‐PCR was able to specifically detect and differentiate GI and GIII JEV. The multiplex RT‐PCR was able to successfully differentiate GI and GIII virus in JEV‐infected mosquitoes. Thus, a sensitive and specific multiplex RT‐PCR system for the rapid detection and differentiation of GI and GIII JEV has been developed, and this test is likely to be valuable when carrying out mosquito‐based JEV surveillance. |
