title: 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 × 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.
authors.affiliation: Division of Veterinary Public Health, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, 243 122, India, hdhanze@rediffmail.com.
authors.forename: H
authors.initials: H
authors.lastname: Dhanze
authors.forename: K N
authors.initials: KN
authors.lastname: Bhilegaonkar
authors.forename: G V P P S
authors.initials: GV
authors.lastname: Ravi Kumar
authors.forename: P
authors.initials: P
authors.lastname: Thomas
authors.forename: H B
authors.initials: HB
authors.lastname: Chethan Kumar
authors.forename: M
authors.initials: M
authors.lastname: Suman Kumar
authors.forename: S
authors.initials: S
authors.lastname: Rawat
authors.forename: P
authors.initials: P
authors.lastname: Kerketta
authors.forename: D B
authors.initials: DB
authors.lastname: Rawool
authors.forename: A
authors.initials: A
authors.lastname: Kumar
mesh_terms: D000818:Animals; D001769:Blood / Q000821:virology*; D004664:Encephalitis Virus, Japanese / Q000302:isolation & purification*; D004672:Encephalitis, Japanese / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis* / Q000821:virology*; D014730:Veterinary Medicine / Q000379:methods*