Resumo

VILLARREAL CAMACHO, José et al. Polimerase chain reaction technique for detecting Salmonella sp. in powdered milk: optimization of the method in 12 hours. Salud, Barranquilla [online]. 2008, vol.24, n.2, pp.216-225. ISSN 0120-5552.

The traditional methods to identify Salmonella sp. are based on the culture medium use that allows the recovery of the micro organism, isolation in selective media, biochemical and serologic characterization. These methods are tedious, have a low specificity and sensitivity and they generally consume a long time. The main objective of this study was to standardize and to optimize the PCR technique to detect Salmonella sp. in 12 hours, from DNA of pure cultures and from powdered milk samples, intentionally inoculated with 200, 20 and 2 CFU/mL. For the extraction of DNA, two methods were used: phenol:chloroform:isoamyl alcohol and chelex® 100. The optimization of the temperature of hibridización and the concentrations of Magnesium Chloride, using an incomplete factorial desing 6x7 allowed to establish a detection limit of up to 10 pg of DNA from pure cultures of Salmonella typhi. The PCR was based on the specificity of oligonucleotidos the 139-141, that amplified a band of 284 pb for the gender identification. The results show that: (I) the inhibitor addition of accompanying flora like Novobiocin (45 mg/L) or brilliant green (10 mg/L) as inhibitors of accompanying flora, after the first three hours in the nonselective pre-enrichment of 6 hours, does not significantly influence in the recovery of the bacterial cells, (II) when obtaining biomass of the first dilution in base 10 and using the phenol:chloroform:isoamyl alcohol technique for the extraction of DNA; can be detected 2 CFU/mL Salmonella s.p. from powdered milk and that the PCR technique reduces the time of test considerably.

Palavras-chave : Salmonella sp.; powdered milk; PCR; molecular diagnosis; microbiologist diagnosis; optimization.

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