Comparative evaluation of nucleic acid-based assays for detection of Japanese encephalitis virus in swine blood samples
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.
KeywordsReverse Transcription Polymerase Chain Reaction West Nile Virus Japanese Encephalitis Virus Japanese Encephalitis Classical Swine Fever Virus
Conflict of interest
The authors declare no conflict of interest.
- 2.Yadav JS (2006) A special issue on Japanese encephalitis. ENVIS News Lett 3:1–11Google Scholar
- 6.OIE (2010) Japanese encephalitis. In: OIE Terrestrial Manual, Chapter 2.1.7, pp. 1-11Google Scholar
- 7.Parida M, Santhosh SR, Dash PK, Tripathi NK, Saxena P, Ambuj S (2006) Development and evaluation of reverse transcription loop-mediated isothermal amplification assay for rapid and real-time detection of Japanese encephalitis virus. J Clin Microbiol 44:4172–4178CrossRefPubMedCentralPubMedGoogle Scholar
- 8.Santhosh SR, Parida MM, Dash PK, Pateriya A, Pattnaik B, Pradhan HK, Tripathi NK, Ambuj S, Gupta N, Saxena P, Rao PVL (2007) Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantitation of Japanese encephalitis virus. J Virol Methods 143:73–80CrossRefPubMedGoogle Scholar
- 9.Krieg P (1991) Improved synthesis of full length RNA probe at reduced incubation temperatures. Nucleic Acids Res 18:643Google Scholar