Evaluation of real-time reverse-transcription loop-mediated isothermal amplification assay for clinical diagnosis of West Nile virus in patients

Abstract

Background & objectives: West Nile virus (WNV) is a mosquito-borne flavivirus. The disease can be diagnosed by isolation followed by fluorescent antibody tests, enzyme-linked immunosorbent assay and polymerase chain reaction (PCR) assay. These diagnostic methods are laborious and time-consuming. The present study was aimed to evaluate the real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, early and accurate diagnosis of WNV.

Methods: A one-step single tube accelerated quantitative RT-LAMP assay was evaluated by targeting the Env gene of WNV. The gene amplification was accomplished by incubating the reaction mixture at 63°C for 60 min in both real time turbidimeter as well as routine laboratory water bath/dry heating bath. To rule out contamination issues, proper negative controls, including no template, no primer; and no enzyme, were always kept alongside each run. The RT-LAMP assay was evaluated on 105 clinical samples from individuals having ocular infection.

Results: Of the 105 samples tested, 27 were positive for WNV by RT-LAMP assay. The comparative evaluation with conventional RT-PCR revealed 100 per cent accordance with sensitivity and specificity of 100 and 95 per cent, respectively. The specificity of this assay was confirmed with serum samples obtained from patients with dengue and chikungunya.

Interpretation & conclusions: The RT-LAMP test seemed to be a sensitive and specific method for rapid detection of WNV infection and would be useful for rapid screening of a large number of clinical samples in endemic areas during outbreaks.

Keywords: Arbovirus; West Nile virus; detection assay; envelope gene; reverse transcription loop-mediated isothermal amplification test and clinical diagnosis.

Fig. 1

Comparison between sensitivity (detection limit) of reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and conventional RT-PCR. (A) Real-time kinetics of West Nile virus reverse-transcription loop-mediated isothermal amplification (RT-LAMP) amplification of the E gene showing the amplification curve with serial 10 fold dilutions of the virus ranging from 10⁴ to 0.0001 plaque-forming unit (pfu). The x axis depicts the time of positivity, and the y axis shows the turbidity value in terms of the optical density at 400 nm. (B) RT-PCR performed on the same serial dilution as used for RT-LAMP. The detection limit of the RT-LAMP assay was 10-fold higher than conventional RT-PCR. M, 1 kb marker; lane1-9, 104 - 0.0001 pfu of virus; lane 10, no virus.

Fig. 2

The optical density profile of different patient samples including healthy controls as obtained through the West Nile-specific loop-mediated isothermal amplification (RT-LAMP) assay. PUO, pyrexia of unknown origin; DEN, dengue; CHIK, chikungunya.