Development and evaluation of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) for avian influenza A and rabies and other lyssaviruses

Abstract

Nucleic acid diagnostic tests can complement existing tools to improve the diagnosis of diseases. However, the requirement of laboratory infrastructure limits their use in developing countries as routine tests. Isothermal amplification techniques such as loop-mediated isothermal amplification bridge such challenges because they require simpler infrastructure. In this study, we evaluated a commercial H5N1 Avian influenza virus (AIV) detection kit using isothermal amplification with Bsm DNA polymerase. We also developed a one-step RT-LAMP assay using two strand displacing DNA polymerases for the detection of two zoonotic viruses, avian influenza A virus and lyssaviruses. During the evaluation of the commercial H5N1 detection kit, A/H9N2, two A/H7N3 isolates and Influenza B were also detected indicating non-specific detection. After optimising reaction temperature and time only the Influenza B was non-specifically detected. In addition, a reverse-transcription LAMP assay was developed focusing on the matrix gene of avian influenza A virus. The detection limit and specificity of the assay was tested using serially diluted in vitro transcribed RNA and the different subtypes of influenza virus using optimal reaction conditions. Restriction enzyme digestion and nucleotide sequencing was used to confirm the identity of the amplified RT-LAMP product. Two detection methods, agarose gel electrophoresis and realtime fluorescence using a fluorescence reader, ESE-Quant tube scanner (ESE Gmbh, Stockach, Germany), were evaluated. The sensitivity of these two detection methods was similar; however, the real-time monitoring of amplification is more suitable for field application of the RT-LAMP assays. A reverse-transcription loop-mediated isothermal amplification assay was also developed for the specific detection of rabies and other lyssaviruses. The assays used specifically designed primers to target the partial nucleoprotein (N) gene and were able to amplify all 12 lyssavirus species; representing a wide diversity of lyssaviruses present in Africa. RT-LAMP reaction was confirmed by restriction enzyme analysis and sequencing. The use of melting curve analysis was also attempted. The assay was about 1000 times more sensitive compared to the RTPCR assay. The RT-LAMP assays described here have great potential as a diagnostic tool as well as an on-site molecular tool especially in resource-limited settings.