Resumen

LEYVA MARTINEZ, Robert M. et al. Nested PCR multiplex assay for detection of phytoplasma and begomoviruses identified in soybean in Cuba. Rev. Protección Veg. [online]. 2019, vol.34, n.2, e07.  Epub 08-Nov-2019. ISSN 1010-2752.

The objective of the work was to develop a nested multiplex PCR assay for the simultaneous detection of phytoplasmas and the begomoviruses identified in soybean in Cuba. Two pairs of specific primers to the begomoviruses Tobacco yellow crinkle virus (TbYCV) and Rhynchosia golden mosaic Yucatan virus (RhGMYuV) were designed, and they were used in conjunction with two pairs of universal primers for the generic detection of phytoplasms under established conditions. The evaluation of the optimal temperature for alignment of the primers to the template, the analytical sensitivity, and the specificity of the primers, critical parameters for the nested PCR assay for begomoviruses, were optimized. Critical parameters for the nested multiplex PCR assay, such as the evaluation of different combinations of concentrations of the primers designed with the conditions established for phytoplasm nPCR, the analytical sensitivity, and the intra and inter-assay repeatability, were also optimized. Performance parameters of the multiplex nPCR assay were determined. In the multiple assay, the fragments expected for TbYCV (988 kb), RhGMYuV (968 kb), and phytoplasms (1250 kb) were successfully amplified, confirming the simultaneous, sensitive and specific detection of these pathogens. The analytical parameters of the trial performance were over 90%. The multiple nested PCR assay here developed allows a sensitive, fast and low-cost detection of phytoplasmas sp. and begomoviruses that affect soybean in Cuba. These results are the first of this type for the country and provide new methodological elements for the validation of plant pathogen detection assays.

Palabras clave : begomoviruses; phytoplasma; multiplex nested PCR; soybean.

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