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

Autor: H. Dhanze, M. Suman Kumar, K. N. Bhilegaonkar, Prasad Thomas, Priscilla Kerketta, D. B. Rawool, Avanindra Kumar, H.B. Chethan Kumar, Sushma Rawat, G.V.P.P.S. Ravi Kumar
Rok vydání: 2015
Předmět:
Zdroj: Archives of Virology. 160:1259-1266
ISSN: 1432-8798
0304-8608
DOI: 10.1007/s00705-015-2385-3
Popis: 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 × 10(5) 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 × 10(6) and 4.8 × 10(9) 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.
Databáze: OpenAIRE
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