Development of an immunomagnetic separation–propidium monoazide–polymerase chain reaction assay with internal amplification control for rapid and sensitive detection of viable Escherichia coli O157:H7 in milk

Abstract

A rapid, reproducible, sensitive and specific combination assay was developed for Escherichia coli O157:H7 comprising sequential immunomagnetic separation (IMS), followed by propidium monoazide (PMA), exclusion by viable cells, internal amplification control (IAC) and polymerase chain reaction (PCR). IMS was used to improve sensitivity, reduce detection time and eliminate false positives. PMA was used to detect viable cells, and IAC was incorporated into the system to eliminate the inhibitors of PCR from the food matrix. In the presence of inhibitors, IAC produced no signal, thereby eliminating false negative results. The results indicated that the IMS cell capture efficiency was about 90% at a concentration of 1 × 106 cfu mL−1 with a detection limit of 2.1 × 102 cfu mL−1 for pure culture. In an unoptimised system, IMS–PMA–PCR with IAC showed a detection limit of 5 × 103 cfu mL−1 in spiked milk over a 240 min assay, faster than conventional methods of detection.