Fast duplex one-step reverse transcriptase PCR for rapid differential detection of West Nile and Japanese encephalitis viruses

Abstract

The aim of this study was to develop a highly sensitive and specific one-step duplex reverse transcriptase PCR (RT-PCR) assay for the simultaneous and differential detection of West Nile (WNV) and Japanese encephalitis (JEV) viruses. The bioinformatic analysis of published sequences of WNV and JEV revealed conserved regions not targeted by previously reported primers. A total of 13 primers were designed based on these regions to detect all of the WNV and JEV lineages and to discriminate between the two viruses by the generation of 482- and 241-bp cDNA products, respectively. The results indicate that single-tube duplex PCR using these primers is a useful technique for the detection and differentiation of WNV and JEV in plasma or brain tissue. The novel duplex RT-PCR described in this study enables the early diagnosis of these two encephalitic flaviviruses. In addition, this technique may be useful as part of a testing regimen for human patients, horses, and other susceptible animal species, as it is rapid (less than 3.5 h from RNA extraction), sensitive, and specific, and it may enable the differential diagnosis of clinical samples.

FIG. 1.

Gel electrophoresis of the uniplex RT-PCR products. JEV is indicated by the PCR product of 241 bp and WNV by the PCR product of 482 bp. Lane M, 1-kb DNA molecular size marker (100-bp DNA ladder; Bioneer); lane J, Anyang300 strain of JEV; lane WN, NY385-99 strain of WNV; lane WB, B956 strain of WNV.

FIG. 2.

Multiplex RT-PCR amplification of WNV and JEV. Lane M, 1-kb DNA molecular size marker (100-bp DNA ladder; Bioneer); lane JEV, Anyang300 strain of JEV; lane WNV, NY385-99 strain of WNV; lanes WNV and JEV, WNV and JEV, respectively, in a single tube.

FIG. 3.

Detection limit of multiplex RT-PCR assay for the detection of JEV (a) and WNV strains NY385-99 (b) and B956 (c) by using primer mixtures designed in this study. Values are PFU per ml. WNV isolates NY385-99 and B956 were detected at a minimum titer of 10^2.2 PFU/ml (corresponding to 10^4.5 copies of RNA) and 10^1.8 PFU/ml (corresponding to 10^3.7 copies of RNA), respectively, while for JEV the sensitivity was 10^2.4 PFU/ml (corresponding to 10^4.1 copies of RNA) in brain homogenate and plasma.