Resumo

NARANJO, Eber; OTERO, Adriana  e  MARTINEZ-ZUBIAUR, Yamila. Optimization of the amplification by PCR of the endoglucanase gene in Ralstonia solanacearum (Smith) Yabuuchi. Rev. Protección Veg. [online]. 2013, vol.28, n.3, pp.211-214. ISSN 1010-2752.

The current classification scheme of the causal agent of the bacterial wilt Ralstonia solanacearum (Smith) Yabuuchi is based on the analysis of the endoglucanase gene sequence of the bacterium. The objective of this paper was to optimize the PCR amplification of this gene. PCR parameters were determined using the OLIGO software version 7.53 for in silico analyses. The effect of cell concentration on amplification efficiency was assessed by using serial dilutions of R. solanacearum suspensions. For the in vitro assays, a two step-PCR protocol was evaluated with the GoTaq® DNA Polimerasa kit. The cell concentration was a critical factor on amplification efficiency with an optimal value of 10⁸ CFU.ml^-1. Time was reduced for the initial denaturalization and final extension phases and hybridization and extension were combined in a single step resulting in a 57 minute shorter-PCR program than the original protocol. The phylogenetic analysis of this gene sequence will allow performing diversity studies of Cuban isolates of R. solanacearum, establishing comparative relationships with isolates of other geographical locations and updating the disease management strategies.

Palavras-chave : Bacterial wilt; in silico; in vitro.

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