Comparative evaluation of nucleic acid-based assays for detection of Japanese encephalitis virus in swine blood samples

Abstract

Japanese encephalitis is an emerging mosquito-borne flaviviral zoonotic disease. The present study was undertaken with the objective of developing rapid and sensitive nucleic-acid-based assays for detection of Japanese encephalitis virus (JEV) in swine blood samples. Three nucleic-acid-based assays, viz., reverse transcription polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), and real-time RT-PCR, were developed and compared in terms of their diagnostic efficacy. All three assays were found to be 100 per cent specific. The minimum detection limit of RT-LAMP and real-time RT-PCR was 12 copies/µl, while RT-PCR could detect 1.2 × 105 copies/µl. On comparison, RT-LAMP and real-time RT-PCR were 4-log more sensitive than RT-PCR. The applicability of the assays was evaluated by screening 135 field swine blood samples, of which 24 (17.77 %) were positive by RT-LAMP and real-time RT-PCR and only six (4.44 %) were positive by RT-PCR. The viral load in swine blood samples ranged between 2 × 106 and 4.8 × 109 copies per ml of blood by real-time RT-PCR. The comparative diagnostic sensitivity and specificity of RT-LAMP vis-à-vis real-time RT-PCR was found to be 100 %, while the sensitivity and specificity of RT-PCR vis-à-vis real-time RT-PCR was found to be 25 % and 100 %, respectively. Thus, the use of RT-PCR may cause the incidence of JEV in the swine population to be underestimated, while the real-time RT-PCR reported here is the test of choice for reference laboratories, and the newly developed one-step RT-LAMP assay will be suitable for field-level testing.

DOI: 10.1007/s00705-015-2385-3

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@article{Dhanze2015ComparativeEO, title={Comparative evaluation of nucleic acid-based assays for detection of Japanese encephalitis virus in swine blood samples}, author={H. Dhanze and K. N. Bhilegaonkar and G. V. P. P. S. Ravi Kumar and P. Thomas and H. B. Chethan Kumar and M. Suman Kumar and S. Rawat and P. Kerketta and D. B. Rawool and A. Kumar}, journal={Archives of Virology}, year={2015}, volume={160}, pages={1259-1266} }