Development and evaluation of a reverse transcription loop-mediated isothermal amplification assay for H3N8 equine influenza virus.

This research investigates a novel technique known as “reverse transcription loop-mediated isothermal amplification” or RT-LAMP, finding it to be both more sensitive and specific than current diagnostic methods for the detection of H3N8 equine influenza virus, the primary cause of equine influenza.

Methodology

• The researchers focused on RT-LAMP, a method of DNA amplification that allows for rapid, simplified testing at a constant temperature, making it potentially more accessible than methods requiring lab-based temperature cycling.
• Specifically, they used this methodology to target the hemagglutinin gene present in the H3N8 subtype of the equine influenza virus (EIV).
• The detection limit of RT-LAMP in this study was one virus particle in a 10^5 dilution. This sensitivity level is significantly higher than standard tests, outperforming the Espline Influenza A&B-N test by a factor of a thousand and a reverse transcription polymerase chain reaction (RT-PCR) – another DNA testing method – by an order of magnitude.

Testing and Results

• The researchers also assessed the specificity of the RT-LAMP test, using it on various equine pathogens and nasal swabs collected from horses that had fever symptoms in 2010, after the EIV had been eradicated in Japan.
• In these tests, the RT-LAMP assay showed no cross-reactivity – it did not give a positive result for other pathogens, indicating a high level of specificity for EIV.
• Comparatively, in a set of 100 nasal swabs from horses during an actual EIV outbreak in 2007, the RT-LAMP test was able to accurately identify EIV in 52 samples, outperforming both the Espline (17 correct detections) and RT-PCR assays (41 correct detections).

Conclusion

• The RT-LAMP test showed high levels of sensitivity; it was able to detect the virus at lower levels than other tests and high specificity; it only provided positive results when the EIV was present.
• Due to the sensitivity, specificity, and relative ease-of-use (it does not require a thermal cycler or gel electrophoresis), the RT-LAMP assay could be particularly useful for lab-based diagnosis of EIV.