Duplex RT-RPA/RPA assay for simultaneous detection of banana streak MY virus and banana bunchy top virus

Abstract

Banana streak MY virus (BSMYV) and banana bunchy top virus (BBTV) are considered as major constraints to banana production globally, severely impacting yield and international trade. Early detection of these viruses is vital for their effective management. In the present study, a sensitive, easy to use rapid duplex Reverse Transcription-Recombinase Polymerase Amplification (RT-RPA) and RPA assay have been developed for the simultaneous detection of BSMYV and BBTV. The assay utilizes a simple template preparation method involving crude leaf extracts prepared in GEB3 extraction buffer, eliminating the need for complex RNA/DNA isolation and expensive reagents. Further, no cross-reactivity was observed with other banana viruses indicating that the primers used were very specific for the detection of BSMYV and BBTV infections. Optimization results revealed 37 °C for 30 min to be the ideal conditions for the simultaneous amplification of the viruses. The developed RPA based assays showed higher sensitivity, detecting both the viruses at extremely low concentrations i.e. at dilutions of up to 10⁻⁶compared to conventional RT-PCR/PCR. Thus, the developed duplex RPA based assays are significant advancement in virus detection for quick detection in resource constrained laboratories and will be highly useful for certification programs, thereby supporting sustainable banana production in India and worldwide.