Universidad y ciencia
versão impressa ISSN 0186-2979
ROCHA-REVILLA, JC et al. Detection of Pantoea stewartii Mergaert, Verdonck & Kersters directly from maize seeds using IMMUNO-PCR. Universidad y ciencia [online]. 2009, vol.25, n.3, pp. 245-252. ISSN 0186-2979.
Immunological and molecular techniques based on DNA have been used for microorganism detection due to their sensitivity, specificity and speed. However, when the identification of bacteria with zero tolerance is required (eg. Pantoea stewartii), molecular and immunological techniques do not offer a 100% reliability. The objective of this study was to combine the ELISA and PCR techniques in order to complement their advantages and efficiently detect bacteria directly on seeds. A maize seed sample imported from the USA and with a high probability of being infected with Pantoea stewartii, and this was verified with ELISA, was used in this study. Having confirmed the presence of the bacterium, it was isolated and identified with microscopical, biochemical, physiological, tobacco hypersensibility, pathogenicity and PCR amplification of rDNA 16S tests. The immuno-PCR technique was then optimised (primer annealing temperature, antibody concentration, incubation temperature and microtube branding). The study showed that it is possible to amplify the DNA of P. stewartii directly from maize seeds, with an annealing temperature of 62 and 65 °C, incubation times of 1-3 hours, antibody concentrations of 5:200 and 10:200 and Fisher and Axigen microtubes. The adaptation and optimisation of the immuno-PCR technique is important as it may aid in the reduction of the risk of P. stewartii propagation towards areas free of bacteria.
Palavras-chave : ELISA; antibodies; microtube brand; Stewart's wilt.