D H D'Souza1, L-A Jaykus. 1. Department of Food Science, North Carolina State University, Raleigh, NC, USA. ddsouza@unity.ncsu.edu
Abstract
AIMS: The purpose of this study was to apply nucleic acid sequence-based amplification (NASBA) for the detection of Salmonella enterica serovar Enteritidis (S. Enteritidis) in representative foods. METHODS AND RESULTS: A previously reported primer and probe set based on mRNA sequences of the dnaK gene of Salmonella were used in this study. To test for possible food matrix inhibition and assay detection limits, 25-g samples of representative food commodities (fresh meats, poultry, fish, ready-to-eat salads and bakery products) were pre-enriched with and without S. Enteritidis inoculation. The NucliSens(R) Basic Kit, supplemented with enzymes from various other commercial sources, was used for RNA isolation, NASBA amplification and electrochemiluminescent (ECL) detection. The end point detection limit of the NASBA-ECL assay was equivalent to 101 CFU of S. Enteritidis per amplification reaction. When the assay was tested on noncontaminated foods, none of the food matrices produced false-positive results. Some of the food matrices inhibited the NASBA-ECL reaction unless the associated RNA was diluted 10-fold prior to amplification. CONCLUSIONS: For all food items tested, positive ECL signals were achieved after 18 h of pre-enrichment and subsequent NASBA at initial inoculum levels of 102 and 101 CFU per 25 g food sample. SIGNIFICANCE AND IMPACT OF THE STUDY: This rapid, semi-automated detection method has potential for use in the food, agricultural and public health sectors.
AIMS: The purpose of this study was to apply nucleic acid sequence-based amplification (NASBA) for the detection of Salmonella enterica serovar Enteritidis (S. Enteritidis) in representative foods. METHODS AND RESULTS: A previously reported primer and probe set based on mRNA sequences of the dnaK gene of Salmonella were used in this study. To test for possible food matrix inhibition and assay detection limits, 25-g samples of representative food commodities (fresh meats, poultry, fish, ready-to-eat salads and bakery products) were pre-enriched with and without S. Enteritidis inoculation. The NucliSens(R) Basic Kit, supplemented with enzymes from various other commercial sources, was used for RNA isolation, NASBA amplification and electrochemiluminescent (ECL) detection. The end point detection limit of the NASBA-ECL assay was equivalent to 101 CFU of S. Enteritidis per amplification reaction. When the assay was tested on noncontaminated foods, none of the food matrices produced false-positive results. Some of the food matrices inhibited the NASBA-ECL reaction unless the associated RNA was diluted 10-fold prior to amplification. CONCLUSIONS: For all food items tested, positive ECL signals were achieved after 18 h of pre-enrichment and subsequent NASBA at initial inoculum levels of 102 and 101 CFU per 25 g food sample. SIGNIFICANCE AND IMPACT OF THE STUDY: This rapid, semi-automated detection method has potential for use in the food, agricultural and public health sectors.