TaqMan real-time RT-PCR assay for detecting Japanese encephalitis virus in swine blood samples and mosquitoes

Abstract

Japanese encephalitis (JE) is an emerging mosquito-borne zoonotic flaviviral disease. The present study was undertaken with the objective to develop TaqMan real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for rapid detection and quantification of Japanese encephalitis virus (JEV) in swine blood and mosquito vectors. The amplification of envelope (E) gene was targeted by designing gene-specific MGB TaqMan fluorescent probe along with the primers. The best performance in terms of sensitivity was achieved by standardized TaqMan real-time RT-PCR with a detection limit of 2.8 copies/reaction and it was found to be 4-log more sensitive than conventional RT-PCR. The applicability of the standardized TaqMan assay was evaluated by screening representative sets of field swine blood samples and mosquito pools for JEV. The viral load ranged between 3.32 × 10⁷–4.2 × 10² copies/ml of swine blood samples, and 5.7 × 10⁹–1.3 × 10² copies/pool of mosquitoes. The standardized assay which is highly sensitive, specific and rapid would aid in screening sentinel swine and mosquitoes under JEV surveillance programs for effective prevention and control of disease in human beings.

KEYWORDS:

• Japanese encephalitis virus
• TaqMan real-time RT-PCR
• swine blood
• mosquito pool

Acknowledgements

We are highly thankful to Dr. Ashwani Kumar, Deputy Director, National Institute of Malaria Research, Goa, India and laboratory staff for providing training on mosquito identification. We are grateful to Indian Council of Agriculture Research-Outreach Program on zoonotic diseases for financial grant to conduct the present study.

Disclosure statement

No potential conflict of interest was reported by the authors.