Development of a bead-based assay for detection of three banana-infecting viruses

Background: Banana bunchy top virus (BBTV), cucumber mosaic virus (CMV) and banana streak virus (BSV) are important banana viruses, there are possible infections frequently with several viruses in field. Since the viruses are readily trasmitted in vegetative propagules, which pose a threat to banana production in banana-growing areas. Methods: A multiplex polymerase chain reaction (PCR) protocol combined with LiquiChip analysis to identify BSV, BBTV, and CMV, with consistent amplification of plant ubiquitin (UBQ), the banana plant messenger RNA used as a procedural control. Multiplex reverse transcription (RT)-PCR amplicons were extended by allele-specific primers, followed by hybridization with carboxylated microspheres containing unique fluorescent oligonucleotides, which were detected using the LiquiChip 200 workstation. Results: In this study, we aimed to develop a rapid, sensitive, and simultaneous detection method for BSV, BBTV, and CMV using a bead-based multiplex assay that can be applied in routine diagnosis. We demonstrated that this detection system was extremely efficient and highly specialized for differentiating individual in a mixture of viruses while being ten times more sensitive than traditional RT-PCR. The development of this method makes it feasible to detect banana viruses in field collected leaf samples.