Development and evaluation of reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) for avian influenza A and rabies and other lyssaviruses
Year: 2012
Type: dissertation
Abstract: Nucleic acid diagnostic tests can complement existing tools to improve the diagnosis \nof diseases. However, the requirement of laboratory infrastructure limits their use in \ndeveloping countries as routine tests. Isothermal amplification techniques such as \nloop-mediated isothermal amplification bridge such challenges because they require \nsimpler infrastructure. In this study, we evaluated a commercial H5N1 Avian \ninfluenza virus (AIV) detection kit using isothermal amplification with Bsm DNA \npolymerase. We also developed a one-step RT-LAMP assay using two strand displacing DNA polymerases for the detection of two zoonotic viruses, avian \ninfluenza A virus and lyssaviruses. \nDuring the evaluation of the commercial H5N1 detection kit, A/H9N2, two A/H7N3 \nisolates and Influenza B were also detected indicating non-specific detection. After \noptimising reaction temperature and time only the Influenza B was non-specifically \ndetected. In addition, a reverse-transcription LAMP assay was developed focusing on \nthe matrix gene of avian influenza A virus. The detection limit and specificity of the \nassay was tested using serially diluted in vitro transcribed RNA and the different \nsubtypes of influenza virus using optimal reaction conditions. Restriction enzyme \ndigestion and nucleotide sequencing was used to confirm the identity of the amplified \nRT-LAMP product. Two detection methods, agarose gel electrophoresis and realtime \nfluorescence using a fluorescence reader, ESE-Quant tube scanner (ESE \nGmbh, Stockach, Germany), were evaluated. The sensitivity of these two detection \nmethods was similar; however, the real-time monitoring of amplification is more \nsuitable for field application of the RT-LAMP assays. \nA reverse-transcription loop-mediated isothermal amplification assay was also \ndeveloped for the specific detection of rabies and other lyssaviruses. The assays \nused specifically designed primers to target the partial nucleoprotein (N) gene and \nwere able to amplify all 12 lyssavirus species; representing a wide diversity of \nlyssaviruses present in Africa. RT-LAMP reaction was confirmed by restriction \nenzyme analysis and sequencing. The use of melting curve analysis was also \nattempted. The assay was about 1000 times more sensitive compared to the RTPCR \nassay. The RT-LAMP assays described here have great potential as a \ndiagnostic tool as well as an on-site molecular tool especially in resource-limited \nsettings.
Author: Khanyisile Mdladla
Language: English
Subfield: Biomedical Engineering
Field: Engineering
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W2793147998