Rapid Detection and Identification of Bacterial Pathogens by Using an ATP Bioluminescence Immunoassay

Abstract

Rapid identification of viable bacterial contaminants in food products is important because of their potential to cause disease. This study examined a method for microbial detection by using a combined ATP bioluminescence immunoassay. Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium were selected as target organisms because of their implication in foodborne illness. Various matrices containing the target cells were examined, including ground beef homogenate, apple juice, milk, and phosphate-buffered saline. Specific antibodies were immobilized on the surface of 96-well plates, and then the sample matrices containing target cells in the wells were incubated. Sample matrix (no cells) was used to establish background. The plates were washed, and the wells were incubated with BacTiter-Glo reagent in Mueller-Hinton II broth. Bioluminescent output was measured with the GloMax 96 luminometer. Signal-to-noise ratios were calculated, resulting in a limit of detection of 104 CFU/ml for both E. coli O157:H7 and Salmonella Typhimurium. The limit of detection for both species was not affected by the presence of nontarget cells. The various sample matrices did not affect signal-to-noise ratios when E. coli O157:H7 was the target. A weak matrix effect was observed when Salmonella Typhimurium was the target. A strong linear correlation was observed between the number of cells and luminescent output over 4 orders of magnitude for both species. This method provides a means of simultaneously detecting and identifying viable pathogens in complex matrices, and could have wider application in food microbiology.