Simultaneous detection of cucumber mosaic virus, tomato mosaic virus and potato virus Y by flow cytometry

• Published in: Journal of virological methods 1997-12, Vol.69 (1), p.137-145
• Main Authors: Iannelli, D, D'Apice, L, Cottone, C, Viscardi, M, Scala, F, Zoina, A, Del Sorbo, G, Spigno, P, Capparelli, R
• Format: Article
• Language: English
• Subjects: Antibodies, Viral - immunology; Biological and medical sciences; Cucumovirus - isolation & purification; Flow Cytometry; Fluorescein; Fundamental and applied biological sciences. Psychology; Generalities. Techniques. Transmission, epidemiology, ecology. Antiviral substances, control; Latex particles; Microbiology; Microspheres; Multiparametric flow cytometry; Multiple virus detection; Phycoerythrin; Phytopathology. Animal pests. Plant and forest protection; Plant Extracts; Plant Leaves - virology; Plant Viruses - isolation & purification; Plant viruses and viroids; Potyvirus - isolation & purification; Reproducibility of Results; Sensitivity and Specificity; Techniques used in virology; Tobamovirus - isolation & purification; Virology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/S0166-0934(97)00149-3

The simultaneous detection is described of cucumber mosaic virus (CMV), potato virus Y (PVY) and tomato mosaic virus (ToMV) by flow cytometry. Extracts from leaves of healthy and CMV or PVY infected plants were incubated with latex particles, each with a diameter of 3 μm. Extracts from ToMV infected or uninfected plants, however, were incubated with particles, each with a diameter of 6 μm. Beads were washed and incubated in succession with primary and secondary antibodies, the latter labeled with phycoerythrin (PE) or fluorescein (FITC). CMV and PVY were distinguished on the basis of the fluorescence emitted by FITC and PE; ToMV was distinguished from CMV and PVY on the basis of the different diameter (6 μm) of the particles on which it was adsorbed. The three viruses were detected also by another approach. Latex particles with a diameter of 3, 6 and 10 μm were separately sensitized with antibodies specific for CMV, PVY and ToMV. An equal number of sensitized particles was mixed and incubated with the plant extracts containing the three viruses and then with anti-CMV, anti-PVY and anti-ToMV antibodies labeled with FITC. The study describes also a virus purification method based on the use of antibody coated latex particles. The method is simple technically and applicable to the purification of large as well as minute amounts of different viruses (CMV, PVY and ToMV).