Development of a Rapid Surveillance System for Ross River Virus in Mosquitoes Through Reverse-Transcription Loop-Mediated Isothermal Amplification (RT-LAMP)

The global rise in arboviral diseases can be attributed to the ongoing effects of climate change. Ross River virus (RRV) is an illustrative example of such diseases, with case reports in Australia experiencing a significant surge since 2020. RRV is transmitted to susceptible species, such as horses and humans, through multiple mosquito vectors, namely Culex annulirostris, Aedes camptorhynchus, and more recently Ae. notoscriptus. This disease is not only endemic to Australia but has caused outbreaks in surrounding countries such as Fiji and Papua New Guinea. Currently, there are no therapeutic regimes or vaccinations available for RRV, leaving public health warning systems and advice relying upon disease prediction and surveillance. Commonly utilised methods, such as predictive modelling, are experiencing challenges resulting from an increased mosquito presence and extreme weather patterns, often yielding inaccurate advice. Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) provided a promising solution to mitigate these challenges and is now considered the gold standard in many Australian states. However, this method must be performed in a laboratory setting and requires expensive machinery, thus rendering it inadequate for resource-poor or rural communities. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) serves as a simple and field-deployable substitute with comparable sensitivities and specificity to RT-qPCR, whilst possessing the ability to provide rapid results within 20 min. This paper describes a novel RRV RT-LAMP assay that can detect RRV in as little as one mosquito, with a limit of detection of 1 × 10⁻⁷ ng/µl (~620 copies/µl) and a clinical sensitivity of 84%. Through the addition of tetramethylammonium chloride (TMAC), our assay achieved a 100% specificity and was able to detect RRV RNA as early as 2 min in crude field samples. The simplistic sampling method coupled with our RRV RT-LAMP assay can provide an in-field and low-cost alternative to current routine surveillance techniques.