A real-time PCR-based microfluidics platform for the detection of Cronobacter sakazakii in reconstituted milks

A fully-integrated real-time PCR-based microfluidics platform was developed for the detection of the emerging foodborne pathogen Cronobacter sakazakii in a synthetic culture medium and in reconstituted milks. Initially, a novel real-time PCR protocol involving chemical lysis of bacterial cells and SYBR green for detecting C. sakazakii was developed. Employing this new real-time PCR methodology with the microfluidics platform allowed the detection of as low as 103 and 104 cfu mL−1 of C. sakazakii in tryptone soy broth (TSB) and reconstituted skim milk (RSM), respectively, within 50 min. Sensitivity was improved to 10 and 102 cfu mL−1 of C. sakazakii in TSB and RSM, respectively, by incorporating cell separation by cationic paramagnetic beads (CPB) before detection with the microfluidics platform. The CPB-microfluidics platform was able to detect as few as 102 cfu mL−1 of C. sakazakii in reconstituted full milk and reconstituted infant formula milk within 70 min.