Development of a Real-Time PCR Assay for Detection and Quantification of Escherichia coli O157:H7 in Apple Juice

Apple cider/juice contaminated with Escherichia coli O157:H7 has been implicated in several foodborne illness outbreaks, but due to the presence of reaction inhibitors, detection by polymerase chain reaction (PCR) is often difficult. The studies presented in this dissertation were conducted to evaluate techniques to improve detection of E. coli O157:H7 in apple juice using a fluorogenic probe-based real-time PCR assay without prior enrichment. Two commercial DNA extraction and purification procedures, GenEluteTM Bacterial Genomic DNA Kit and PrepMan® Ultra Sample Preparation Reagent, were combined with two real-time PCR chemistries, SYBR® Green I dye and TaqMan® probes for potential use in the apple juice assay. After real-time PCR, no significant differences were observed in cycle threshold values (Ct) (p>0.05) among the methods. The PrepMan/TaqMan method was subsequently combined with a real-time PCR assay based on detection of the stx1, stx2, and uidA genes. Apple juice was inoculated individually with nine strains of E. coli O157:H7 (1.0 log CFU/ml to 4.0 log CFU/ml) and plated to verify initial inocula. For particulate removal, apple juice was vacuum filtered twice (Whatman No. 4 and Whatman No. 1), followed by a distilled water wash. Samples were plated again to obtain post-filtration inocula. Filtered juice was centrifuged, pellets were resuspended in 1 ml phosphate buffer, and E. coli O157:H7 cells were concentrated by immunomagnetic separation. PrepMan Ultra was added to the magnetic bead/E. coli O157:H7 complex for DNA extraction. Extracts were combined as appropriate with primers, probe and other reagents, and real-time PCR was performed. Average E. coli O157:H7 inoculum levels of 0.3, 2.2, 3.3 and 4.3 log CFU/ml in apple juice were detected at average Ct values of 41.22, 37.54, 34.69 and 31.81 (stx1); 43.13, 38.74, 35.21 and 32.58 (stx2); and 44.13, 41.54, 37.81 and 34.06 (uidA). Across all E. coli O157:H7 strains, populations as low as 1.6 (44 CFU/ml) (stx1), 1.6 (43 CFU/ml) (stx2), and 1.5 (33 CFU/ml) (uidA) log CFU/ml could be quantified using the cell concentration/real-time PCR assay. However, E. coli O157:H7 was detected on occasion below the quantifiable level at the lowest inoculum level for all strains. This method can be used for rapid detection and quantification (<5 h) of E. coli O157:H7 in apple cider/juice and potentially other foods.