Precise, Sensitive Detection of Viable Foodborne Pathogenic Bacteria with a 6-Order Dynamic Range via Digital Rolling Circle Amplification

The presence of viable pathogenic bacteria in food can lead to serious foodborne diseases, thus posing a risk to human health. Here, we develop a digital rolling circle amplification (dRCA) assay that enables the precise and sensitive quantification of viable foodborne pathogenic bacteria. Directly targeting pathogenic RNAs via a ligation-based padlock probe allows for precisely discriminating viable bacteria from dead one. The one-target-one-amplicon characteristic of dRCA enables high sensitivity and a broad quantitative detection range, conferring a detection limit of 10 CFU/mL and a dynamic range of 6 orders. dRCA can detect rare viable bacteria, even at a proportion as low as 0.1%, which is 50 times more sensitive than the live/dead staining method. The high sensitivity for detecting viable bacteria accommodates dRCA for assessing sterilization efficiency. Based on the assay, we found that, for pasteurization, slightly elevating the temperature to 68 °C can reduce the heating time to 10 min, which may minimize nutrient degradation caused by high-temperature exposure. The assay can serve as a precise tool for estimating the contamination by viable pathogenic bacteria and assessing sterilization, which facilitates food safety control.