Development of reverse transcriptase‑recombinase polymerase amplification (RT‑RPA) assay for rapid detection of large cardamom chirke virus

Abstract

Large cardamom chirke virus (LCCV) causing chirke disease of large cardamom is a major production constraint of this crop. Rapid and accurate detection of LCCV is important for managing the disease. In the present study an isothermal assay namely, reverse transcriptase-recombinase polymerase amplification (RT-RPA) was developed for the detection of LCCV. Total RNA isolated by two different methods and crude extracts isolated using five different methods as templates were assessed for their ability to detect LCCV. Of these, only the total RNA isolated by both methods gave consistent and repeatable results while all the crude extracts used as templates gave non-specific amplification. RT-RPA was up to 1000 times more sensitive than conventional RT-PCR for the detection of LCCV. The detection limit of RPA was 10 fg when recombinant plasmid was used as the template. The RT-RPA assay was validated using field samples and found suitable for large-scale screening of large cardamom plants against LCCV for the selection of virus-free plants.