RPA and plant pathogens
TwistAmp® exo RT is being harnessed by researchers in the UK to develop a rapid, field-based diagnostic tool that will help farmers in West Africa to ensure that yam, one of the region’s most important staple food crops, can be propagated free from the most economically important viral disease, Yam mosaic virus (YMV).
With funding from the Bill & Melinda Gates Foundation, Gonçalo Silva, Susan Seal and colleagues at the University of Greenwich’s Natural Resources Institute (NRI) have developed a prototype RT-exoRPA assay for YMV, which has been evaluated to detect YMV in both total RNA extracted from yams and in crude yam sap1.
Carried out at an optimal temperature of 37˚C, the RT-exo RPA assay was found to be as sensitive as RT-PCR, with a detection limit of 14 pg/microliter (purified RNA), and provided results in just 30 minutes. In contrast, it took more than 150 minutes for the RT-PCR assay to reach the same detection sensitivity. “The RT exo RPA assay was also more sensitive than antibody-based tests, which typically require a high level of infection,” comments Dr. Silva. “The closed system that we have designed using the TwistAmp exo kit negates contamination issues, and the fluorescence-based results are viewed in real time. In addition, the 37˚C working temperature is lower than that required by other isothermal methods, such as LAMP (loop-mediated isothermal amplification). Encouragingly, the RPA-based plant pathogen assay worked well, albeit slower and with slightly lower sensitivity, at a temperature of 30˚C, which is a fairly typical ambient temperature in West Africa, so the test could feasibly be carried out without any requirement for heating.”
The NRI team is now working to transfer the RPA-based methodology into a field format that could be used by farmers, propagation sites and certification laboratories to verify that vegetatively propagated seed yam stocks are not infected with YMV. In its published paper, the researchers envisage using battery-operated portable devices for heating the reaction and reading the RPA fluorescence results at the optimum working temperature of 37˚C. “We are now evaluating the fluorescence-based test in microfluidic devices, along with lateral flow-based formats,” Professor Seal states. “The RPA technology is key to our development of a rapid, sensitive and easy-to-use assay that will require minimal equipment and operator steps, and which could help increase yields of this critical food and income crop in West Africa.”
1.Rapid and specific detection of Yam mosaic virus by reverse-transcription recombinase polymerase amplification - Silva G, Bömer M, Nkere C, Kumar PL, Seal SE