Novel Automated Workflow Reducing Time to Result for Detecting O157:H7 and Non-O157 STEC Strains from 375 g Beef Samples

Introduction: Rapid detection of low levels of pathogenic microorganisms in food by molecular methods is often difficult due to the limited amount of targets, complexity of food matrices and presence of PCR inhibitors. We have developed a new workflow for detection of E. coli O157:H7 and 6 non-O157 STECs in 375 g beef samples in less than 8 hours of total Time-to-Result (TTR). The workflow includes an automated sample preparation method and rapid real-time PCR detection of O-group specific genetic targets as well as stx-1/stx-2 and eae genes.

Purpose: To design an effective automated workflow for detecting at least 1 CFU of pathogenic E.coli in 375 g of ground beef and beef trim samples in one 8 hrs working shift.

Methods: 375 g ground beef and beef trim samples were spiked with ~1 CFU of E. coli O157:H7 and non-O157 STECs. After 6.5 hours of enrichment in optimized culture conditions, samples were processed using fast Immuno Magnetic Separation (IMS) sample preparation protocol automated on the MagMAX™ Express-96 Magnetic Particle Processor. The qPCR analysis of purified samples was performed on 7500 Fast Real-Time PCR System under fast cycling conditions. The results of detection were confirmed using plating and overnight enrichment of same samples followed by analysis with AOAC-validated protocols.

Results: 100% correlation was obtained between real-time PCR results and control methods. The total time of the workflow was slightly under 8 hours. The sample preparation method was highly efficient in capturing of target microorganisms and removal of PCR inhibitors, as demonstrated by short TTR and robust detection of the internal positive control included in the assay.  The optimized workflow has minimal number of liquid handling steps that reduces the risk of operator error and cross contamination between samples.

Significance: The ease of use, rapidity and efficiency of our novel IMS-based sample preparation workflow in combination with real-time PCR allow detecting low amounts of pathogenic E.coli strains in difficult food matrices such as 375 g of ground beef in a reduced timeframe.