Rapid Detection of Bacillus cereus by Immunomagnetic Separation and Real-time PCR

Introduction: Bacillus cereus is a rod-shaped and Gram-positive food pathogen, and has been described to cause diarrhea and emetic types of food poisoning. The immunomagnetic separation (IMS) method followed by real-time PCR has been used to capture cells and decreased the assay time which could help to minimize the possibility of exposing consumers to foodborne pathogen.

Purpose: The purpose of this study was to develop a method rapid, specific and quantitative method to detect B. cereus in lettuce and spinach.

Methods: Immunoaffinity magnetic beads were prepared by coating anti-B. cereus antibody to Dynabeads® protein G. The capture kinetics and capacity of the anti-B. cereus antibody against B. cereus were studied between anti-B. cereus antibody and Dynabeads® protein G for concentrations (4, 5, 6, 8 and 10 μg/20 μl), five immunoreactions times (10, 20, 30, 40, 50 min) and a serial of concentrations (102 was carried out using two B. cereus strains and 6 non-target bacteria. The standard curve was constructed using the DNA isolated from the serial dilutions of B. cereus.

Results: The IMBs prepared by 5 μg antibody per 20 μl Dynabeads® were optimized and with the immunoreactions time of 20 min the colony number in the suspension reached the highest. For the specificity assay, B. cereus (2.35 and 2.63 log CFU/100 ml) could be recovered by the IMBs, but for non-target bacteria, less than 1.5 log CFU/100 μl were recovered except for the B. subtilis (2.34 log CFU/100 μl) which showed similar recovery with B. cereus. When combined with the real-time PCR, B. cereus was detected in lettuce and spinach samples with B. cereus > 103 CFU/ml.

Significance: The developed method using IMS combined real-time PCR was rapid, specific and quantitative for detection of B. cereus.