A bead-based suspension array for the multiplexed detection of begomoviruses and their whitefly vectors

Bead-based suspension array systems enable simultaneous fluorescence-based identification of multiple nucleic acid targets in a single reaction. This study describes the development of a novel approach to plant virus and vector diagnostics, a multiplexed 7-plex array that comprises a hierarchical set of assays for the simultaneous detection of begomoviruses and Bemisia tabaci, from both plant and whitefly samples. The multiplexed array incorporates genus, species and strain-specific assays, offering a unique approach for identifying both known and unknown viruses and B. tabaci species. When tested against a large panel of sequence-characterized begomovirus and whitefly samples, the array was shown to be 100% specific to the homologous target. Additionally, the multiplexed array was highly sensitive, efficiently and concurrently determining both virus and whitefly identity from single viruliferous whitefly samples. The detection limit for one assay within the multiplexed array that specifically detects Tomato yellow leaf curl virus-Israel (TYLCV-IL) was quantified as 200fg of TYLCV-IL DNA, directly equivalent to that of TYLCV-specific qPCR. Highly reproducible results were obtained over multiple tests. The flexible multiplexed array described in this study has great potential for use in plant quarantine, biosecurity and disease management programs worldwide.