Sensibilidade do método de obtenção das células bacterianas e da técnica de PCR para detecção de Curtobacterium flaccumfaciens pv. flaccumfaciens em sementes de feijão

Abstract

Currently, there is a need to develop sensitive methods, inexpensive, reproducible and rapid detection of phytobacteria seeds. The objective of this study was to optimize a method for obtaining bacterial cells and the PCR technique to detect Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), in bean seeds using PCR. It was used one pairs of primers CffFOR2-REV4, designed from an PCR amplified fragment of the conserved repetitive sequence (Rep-PCR). The primer pair CffFOR2-REV4 was selected in this study, because demonstrated higher reproducibility and efficacy in the detection of Cff strains in bean seeds. Four methods were also evaluated in the seed extract preparation for PCR: 1) rough seed extract; 2) millipore membrane filter (0.22Mu diameter) concentrated seed extract and subsequently ressuspended in water; 3) centrifuge concentrated seed extract 10.000 xg for 15 minutes for 20 and 80 mL; 4) Bio-PCR. Among those methods, either Bio-PCR or centrifuge concentrated extract, in a total 20 or 80 mL suspension volume, produced a 306bp DNA fragment, diagnostic for Cff. Those two techniques detected the bacterium and presented high sensitivity, detecting up to 1 Cff physiological conditioning artificially contaminated seed in a total 999 healthy ones. A total of seventeen commercial bean seed lots were analyzed by the method of concentration of the extract by centrifugation, and twelve detected in the bacterium Cff, observed by the presence of bands with 306 bp. Therefore, it was possible to optimize a method of obtaining the bacterial cells and PCR for detection of Cff in bean seeds, that is sensitive, reproducible and easy to perform, which can be used routinely in laboratories for seed.