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.
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References
Campbell GL, Hills SL, Fischer M, Jacobson JA, Hoke CH, Hombach JM, Marfin AA, Solomon T, Tsai TF, Tsu VD, Ginsburg AS (2011) Estimated global incidence of Japanese encephalitis: a systematic review. Bull World Health Organ 89:766–774E
Yadav JS (2006) A special issue on Japanese encephalitis. ENVIS News Lett 3:1–11
Guerin B, Pozzi N (2005) Viruses in boar semen: detection and clinical as well as epidemiological consequences regarding disease transmission by artificial insemination. Theriogenology 63:556–572
Liu H, Liu ZJ, Jing J, Ren Q, Liu YY, Guo H, Fan M, Lu HJ, Jin NY (2012) Reverse transcription loop-mediated isothermal amplification for rapid detection of Japanese encephalitis virus in Swine and mosquitoes. Vector Borne Zoonotic Dis 12:1042–1052
Erlanger TE, Weiss S, Keiser J, Utzinger J, Wiedenmayer K (2009) Past, present, and future of Japanese encephalitis. Emerg Infect Dis 15:1–7
OIE (2010) Japanese encephalitis. In: OIE Terrestrial Manual, Chapter 2.1.7, pp. 1-11
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 44:4172–4178
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 143:73–80
Krieg P (1991) Improved synthesis of full length RNA probe at reduced incubation temperatures. Nucleic Acids Res 18:643
Fulmali PV, Sapkal GN, Athawale S, Gore MM, Mishra AC, Bondre VP (2011) Introduction of Japanese encephalitis virus genotype I, India. Emerg Infect Dis 17:319–321
Igarashi A, Tanaka M, Morita K, Takasu T, Ahmed A, Ahmad A, Akram DS, Waqar MA (1994) Detection of West Nile and Japanese encephalitis viral genome sequences in cerebrospinal fluid from acute encephalitis cases in Karachi, Pakistan. Microbiol Immunol 38:827–830
Lian WC, Liau MY, Mao CL (2002) Diagnosis and genetic analysis of Japanese encephalitis virus infected in horses. J Vet Med B Infect Dis Vet Public Health 49:361–365
Paranjpe S, Banerjee K (1998) Detection of Japanese encephalitis virus by reverse transcription/polymerase chain reaction. Acta Virol 42:5–11
Chen Z, Liao Y, Ke X, Zhou J, Chen Y, Gao L, Chen Y, Yu S (2011) Comparison of reverse transcription loop-mediated isothermal amplification, conventional PCR and real-time PCR assays for Japanese encephalitis virus. Mol Biol Rep 38:4063–4070
Toriniwa H, Komiya T (2006) Rapid detection and quantification of Japanese encephalitis virus by real-time reverse transcription loop-mediated isothermal amplification. Microbiol Immunol 50:379–387
Mackay IM, Arden KE, Nitsche A (2002) Real-time PCR in virology. Nucleic Acids Res 30:1292–1305
Yang DK, Kweon CH, Kim BH, Lim SI, Kim SH, Kwon JH, Han HR (2004) TaqMan reverse transcription polymerase chain reaction for the detection of Japanese encephalitis virus. J Vet Sci 5:345–351
Pang X, Lee B, Chui L, Preiksaitis JK, Monroe SS (2004) Evaluation and validation of real-time reverse transcription-PCR assay using the LightCycler system for detection and quantitation of norovirus. J Clin Microbiol 42:4679–4685
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Dhanze, H., Bhilegaonkar, K.N., Ravi Kumar, G.V.P.P.S. et al. Comparative evaluation of nucleic acid-based assays for detection of Japanese encephalitis virus in swine blood samples. Arch Virol 160, 1259–1266 (2015). https://doi.org/10.1007/s00705-015-2385-3
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DOI: https://doi.org/10.1007/s00705-015-2385-3