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.
