Rapid and absolute quantification of VBNC Cronobacter sakazakii by PMAxx combined with single intact cell droplet digital PCR in infant foods

Cronobacter sakazakii (C. sakazakii) in viable but nonculturable (VBNC) state might evade traditional culture detection, and pose a serious threat to food safety. The objective of this study was to establish an improved propidium monoazide (PMAxx), combined with a single intact cell droplet digital PCR (SIC ddPCR) method for rapid and absolute quantification of VBNC C. sakazakii and apply it to the detection of several infant foods. In this study, we optimized the detection conditions for C. sakazakii, and determined the optimal PMAxx final concentration (8 μM), light exposure times (10 min), annealing temperature (58 °C) and elongation time (1 min). In addition, PMAxx-SIC ddPCR demonstrated a higher detection sensitivity and accuracy relative to real-time PCR (qPCR), with 23 CFU/mL in pure culture. Furthermore, PMAxx-SIC ddPCR was used to detect VBNC C. sakazakii in the actual infant food samples. A total of 240 samples were analyzed, out of which 6 samples turned out to be positive by the traditional culture method, whereas 11 samples were found as positive by PMAxx-SIC ddPCR. These results demonstrate that the presence of VBNC may cause the conventional culture-based method to underestimate the size of microbial population.