Multiplex detection of three banana viruses by reverse transcription loop-mediated isothermal amplification (RT-LAMP)

Banana bunchy top virus (BBTV), banana streak viruses (BSVs) and cucumber mosaic virus (CMV) are frequently reported infecting bananas globally. Effective control of their spread depends on robust detection of these viruses in propagation stock, planting material, infected nursery plants, and through strict quarantine. We developed single reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays for BBTV, banana streak OL virus (BSV-OL) and CMV that were sensitive, specific, efficient, and completed in less than 60 min. RNA-based RT-LAMP minimized false positives that arose from banana genomes harboring endogenous viral genomes, such as BSVs. RT-LAMP was also more sensitive than RT-PCR in detecting the DNA viruses, BBTV and BSV-OL, in infected plants. We also developed a multiplex assay using three sets of primers specific for each virus to simultaneously detect BBTV, BSV-OL and CMV in a sample of RNA from the same plant. The reliability and convenience of this assay makes it useful for plant quarantine and indexing plants for propagation.