High-throughput detection of spore contamination in food packages and food powders using tiered approach of ATP bioluminescence and real-time PCR

A rapid and quantitative method for detection of Bacillus spores in food/non-alcoholic beverage packages and food powders has been developed using filtration-based ATP bioluminescence and real-time PCR, targeting the sporulation gene (spo0A). In combination with heat activation, the presence and amount of viable bacterial spores (i.e., Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus thuringiensis) was determined within 20 min through ATP signal amplifications. The detection limits of heat activation-ATP bioluminescence assay for B. amyloliquefaciens and B. licheniformis spores on food packages were 1.4 × 102 and 1.0 × 103 CFU/cm2, respectively. In contaminated food powders, B. thuringiensis spores could be detected by the ATP assay within the range of 7.9 × 100 to 3.2 × 104 CFU/mg powder while the PCR detection limit was 614 CFU/mg. Linear relationships between luminescent signal (RLU/mg) and plate count (CFU/mg) were found. The same sample after heat activation-ATP assay could be directly used for real-time PCR as a streamlined detection to confirm the identity of Bacillus spores in food packages and food powders even though some bacterial DNA loss was observed. This tiered approach, filtration-based one-tube ATP luminescence method as a rapid, viable screening and using real-time PCR as confirmation, could serve as a high-throughput tool for the detection of Bacillus spores in the food and beverage industry.

► A high-throughput two-tier detection method for Bacillus spores was developed. ► Viable Bacillus spores are first screened within 20 min via heat-ATP luminescence. ► Real-time PCR assay was developed by targeting spo0A gene of 3 Bacillus species. ► The streamlined real-time PCR assay uses the same sample after the first screening. ► This approach allows rapid and sensitive spore detection in powders and packaging.