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

Determination of microbial load for different beverages and foodstuff by assessment of intracellular ATP

“…In fact, it contributes to the background luminescence and could affect the luciferase reaction in term of signal level and signal stability (Yan et al, 2012). Finally, Ratphitagsanti, Park, Lee, Wu, and Lee (2012) stated that with an established standard curve from ATP bioluminescence assay, estimation of viable spore numbers in food packages and food powders could be obtained, with a detection limit of respectively 50 CFU/mg and 100 CFU/ cm 2 spores detected in real time.…”

“…Rapid identification of viable Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium was obtained by using a combined ATP bioluminescence immunoassay in various matrices containing the target cells, including ground beef homogenate, apple juice and milk (Hunter & Lim, 2010). ATP bioluminescence has been also recently used to detect spores in food packages and food powders in real time (Ratphitagsanti et al, 2012).…”

“…This instrument has a luminescence sensitivity of 10 amol ATP. Ratphitagsanti et al (2012) developed a rapid and quantitative method for detection of Bacillus spores in food/nonalcoholic beverage packages and food powders using Profile 1 Ò ATP assay (New Horizons Diagnostics, MD, USA), a filtration-based ATP luminescence technique, combined with nutrient germinant and heat activation. The Profile 1 Ò Bioluminometer showed a broad range of sensitivity to detect the presence of ATP from 0.009 pg/ mL to 9 ng/mL, and a good correlation between RLU and CFU/mL.…”

“…Bacterial quantification using qPCR can provide higher level of specificity; however, cost, overestimation of cells, environmental inhibitors, and need of a trained expert can limit its broad use. ATP technology on the other hand requires little skill to implement and is field-applicable and if needed, leftover supernatants from IMS/ATP can be saved and used later for qPCR analysis (Ratphitagsanti et al, 2012). Another alternative to increase specificity can be the use of IMS coupled with ATPmeasurement, which allow to specifically measure ATP from the targeted microorganisms (Tu, Patterson, Uknalis & Irwin 2000).…”

Bioluminescence assay for cell viability

“…By way of illustration, if the concentration of microbial ATP is less than 0.6 pmol/ml, the sample contains up to 5·10 4 CFU/ml, and if above 70 pmol/mlmore than 4·10 6 CFU/ml. A combined method for determination of spore con tamination of food products was suggested in [70]. In the first step of the analysis, the authors used biolumines cence ATP assay in the sample: the sample was concen trated in a Filtravette™, somatic cells were disrupted by treatment with a mild lytic agent, and both somatic and free ATP was removed.…”