Development of immunological and molecular methods for detection of salmonellae in foods / Desenvolvimento de métodos imunológicos e moleculares para detecção se salmonelas em alimentos

AUTOR(ES)
DATA DE PUBLICAÇÃO

2005

RESUMO

Salmonella is a major cause of food-borne illness all over the world. Conventional cultural method for detection of this bacterium in foods is laborious and time-consuming, requiring at least 4 days to obtain negative results, and 6–7 days for identification and confirmation of positive samples. In this manner, rapid methods, such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and immunomagnetic separation (IMS), for the detection of salmonellae in foods have been developed and commercialized. However, kits commercially available in Brazil are all imported and have a high cost per sample tested. For these reasons, this work on development of immunological and molecular methods for detection of salmonellae in foods was started. For development of immunological methods, ten monoclonal antibodies (MAbs) specific for an outer membrane protein (OMP) of serogroup B salmonellae were produced and characterized. The MAbs were used as capture and detection antibody in a sandwich ELISA specific for detection of S. Typhimurium in meats. Detection limit of the sandwich ELISA was 10 5 CFU/ml and it was able to detect 1-10 CFU/25 g of beef and chicken samples experimentally contaminated. The total time of the assay was 57 h. Moreover, a reagent composed of magnetic beads and MAb for use in IMS was developed. This reagent was able to separate and concentrate efficiently S. Typhimurium from pure culture and artificially contaminated meat. For development of the molecular method PCR, a salmonellae-specific sequence of nucleotides (gene fimA) was selected, primers were designed and an internal amplification control (IAC) was developed. This PCR presented 100% of sensibility and specificity and detection limit of 10 4 CFU/ml of salmonellae and 1,5 ρ g of DNA for reaction. The reagent for IMS and the PCR for amplification of gene fimA were associated with success in the development of a novel method for detection of S. Typhimurium in meats. The detection limit of this method from pure culture was 10 4 CFU/ml and from artificially contaminated meat, 1- 10 CFU/25 g. The total time of the assay was 26 h.

ASSUNTO(S)

microbiologia de alimentos salmonella typhimurium alimentos biotecnologia salmonella desenvolvimento de pcr separação imunomagnética anticorpos monoclonais

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