Reverse transcription loop-mediated isothermal amplification (rt-lamp) assay for the detection of rabies virus

Abstract

A one-step real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting rabies virus (RABV) genome. RT-LAMP primers were designed based on the most conserved region of N gene sequences covering various rabies virus isolates of India and representative sequences from each genetic lineage of rabies virus around the world. Degenerate bases were included in the RT-LAMP primers depending on the sequence variation among the isolates. Amplification products of the RT-LAMP assay was monitored by realtime turbidimeter, agarose gel electrophoresis and visual colour change of HNB dye. Detection limit of the RT-LAMP assay was 103 RNA copies or 10-1 FFU (0.1 Fluorescence focus units) of rabies virus particles per reaction. A total of 45 known rabies positive clinical tissues were tested using the RT-LAMP assay and all these samples were confirmed as rabies by all the three detection methods of RT-LAMP assay. Both diagnostic specificity and sensitivity of the assay were 100% comparable to RT-PCR. Thus this technique is an excellent tool for diagnosis and epidemiological studies of rabies, where the disease is enzootic which can be carried out with minimum laboratory facilities.