Traditional methods of identifying food-borne pathogens are time consuming and laborious, so innovative methods for their rapid identification must be developed. Testing for bioluminescence pyrophosphate is a convenient and fast method of detecting pathogens without complex equipment. However, the sensitivity of the method is not as high as that of other methods, and it has a very high detection limit. In this study, the method was optimized to improve its sensitivity. The shortcomings of the method were first identified and corrected using dATPαS instead of dATP for the polymerase chain reaction (PCR), therefore reducing the background signal. Also, when the DNA template extracted from the food-borne pathogens was purified, the new bioluminescence pyrophosphate assay had a limit of detection of <10 copy/μl or 10 colony-forming units/ml, and its sensitivity was higher than that of fluorescent real-time quantitative PCR. Moreover, a single copy of a food-borne pathogen could be detected when a single DNA template was included in the PCR. Salmonella was detected in and isolated from 60 samples of broiler chicken, and the accuracy of the results was verified using a culture method (GB 4789.4-2010). These results showed that the new bioluminescence pyrophosphate assay has the advantages of an intuitive detection process, convenient operation, and rapid measurements. Therefore, it can be used for the rapid detection of pathogenic bacteria and probiotics in various fields.
Keywords: food application; newly bioluminescence pyrophosphate assay; pathogens; qPCR.
© 2019 John Wiley & Sons, Ltd.