Development of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) assays for the detection of two novel viruses infecting ginger

Abstract

Our recent studies have shown the association of two novel viruses namely, ginger chlorotic fleck-associated virus 1 (GCFaV-1) and ginger chlorotic fleck-associated virus 2 (GCFaV-2) with chlorotic fleck disease of ginger. As ginger is propagated through vegetative means, the development of diagnostics would aid in the identification of virus-free plants. In the present study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) assays were developed and validated for the quick detection of GCFaV-1 and GCFaV-2. The detection limits of viruses by these assays, when compared with conventional and real-time RT-PCR, showed that RT-LAMP was up to 1000 times more sensitive than conventional RT-PCR and one-hundredth that of real-time RT-PCR for both the viruses. The detection limit of RT-RPA for GCFaV-1 was up to 100 times more than that of RT-PCR and one-thousandth that of real-time RT-PCR. On the other hand, for detecting GCFaV-2, RT-RPA was found up to 1000 times more sensitive than conventional RT-PCR and one hundredth that of real-time RT-PCR. Based on the cost-effectiveness and duration, RT-LAMP and RT-RPA assays can be suggested for the rapid detection of both viruses