Abstract
BackgroundIris yellow spot virus (IYSV) is an Orthotospovirus that infects most Allium species. Very few approaches for specific detection of IYSV from infected plants are available to date. We report the development of a high-sensitive Luminex xMAP-based microsphere immunoassay (MIA) for specific detection of IYSV.ResultsThe nucleocapsid (N) gene of IYSV was cloned and expressed in Escherichia coli to produce the His-tagged recombinant N protein. A panel of monoclonal antibodies (MAbs) against IYSV was generated by immunizing the mice with recombinant N protein. Five specific MAbs (16D9, 11C6, 7F4, 12C10, and 14H12) were identified and used for developing the Luminex xMAP-based MIA systems along with a polyclonal antibody against IYSV. Comparative analyses of their sensitivity and specificity in detecting IYSV from infected tobacco leaves identified 7F4 as the best-performed MAb in MIA. We then optimized the working conditions of Luminex xMAP-based MIA in specific detection of IYSV from infected tobacco leaves by using appropriate blocking buffer and proper concentration of biotin-labeled antibodies as well as the suitable ratio between the antibodies and the streptavidin R-phycoerythrin (SA-RPE). Under the optimized conditions the Luminex xMAP-based MIA was able to specifically detect IYSV with much higher sensitivity than conventional enzyme-linked immunosorbent assay (ELISA). Importantly, the Luminex xMAP-based MIA is time-saving and the whole procedure could be completed within 2.5 h.ConclusionsWe generated five specific MAbs against IYSV and developed the Luminex xMAP-based MIA method for specific detection of IYSV in plants. This assay provides a sensitive, high-specific, easy to perform and likely cost-effective approach for IYSV detection from infected plants, implicating potential broad usefulness of MIA in plant virus diagnosis.
Highlights
Iris yellow spot virus (IYSV) is an Orthotospovirus that infects most Allium species
The results showed that the rabbit antibody-coated microsphere and SA-RPEcoupled 7F4 had high median fluorescent intensity (MFI) value to sample and low MFI value to buffer control, while the monoclonal antibodies (MAbs) 11C6 and 12C10 followed in the performance
We found that the microsphere immunoassay (MIA) system could only detect the leaf samples infected by IYSV, but not those infected by other six viruses (Table 2), indicating that the MIA method we developed has high specificity in detecting IYSV
Summary
Iris yellow spot virus (IYSV) is an Orthotospovirus that infects most Allium species. Very few approaches for specific detection of IYSV from infected plants are available to date. Iris yellow spot virus (IYSV) is an Orthotospovirus in the family Tospoviridae of the order Bunyavirales, which has been reported to infect most Allium species such as onion crops and some ornamentals such as Spiny Sowthistle, Irises and Lisianthus [1,2,3]. Diamondshaped lesions are pronounced on infected scapes and gradually merge along the disease progresses, eventually leading to the lodging of infected scapes. In seed crops, this would lead to a severe reduction in yield and quality. Two thrips species, Frankliniella fusca and Thrips tabaci
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