Validation of Two New Real-time PCR Multiplex Kits for the Detection of Shiga Toxin-producing Escherichia coli (STEC)

Introduction:  Shiga toxin-producing Escherichia coli (STEC) are pathogens causing severe foodborne illnesses. Although E. coli O157:H7 strain was first declared as an adulterant in raw beef products, the six non-O157 STEC, O26, O111, O103, O121, O45, and O145 were recently recognized by the Centers for Disease Control and Prevention as being responsible for nearly 2/3 of all STEC infections, leading them to be declared as adulterants in beef by the United States Department of Agriculture.  This USDA regulation is to be enforced starting June 4, 2012.

Purpose:  The purpose of this study was to validate two real-time PCR multiplex kits for the detection of the top 6 non-O157 STEC against the USDA MLG 5B.01 reference method.

Methods:  The two real-time PCR kits form a complete solution in compliance with the MLG 5B.01 recommendations. iQ-Check STEC VirX allows DNA extraction from bacterial enrichment and detection of the stx and eae virulence genes. iQ-Check STEC SerO can then be used to screen specifically for the presence of the 6 non-O157 serogroups plus O157:H7.  Inclusivity/exclusivity of the method was tested with 230 E. coli strains of 165 different O-groups, and 31 non-E. coli strains.  The sensitivity of the iQ-Check STEC method was compared to that of the MLG 5B.01 method on artificially contaminated matrices.  Beef trim samples (375g or 325g) were spiked with low levels (2-10 CFU) of an STEC strain and stored at 4°C for 48h. Samples were subsequently analysed in parallel by both methods, A fractional recovery study, involving 50 samples  was carried out for comparison of detection limits.

Results:   Presence or absence of stx (except for the stx2f subtype), eae, and top 6 serogroups were correctly determined for all 230 strains tested.  There was no significant difference in the performance of the iQ-Check method when compared to the MLG 5B.01 reference method for detection of low levels of STEC in beef trim.  The fractional recovery study demonstrated a comparable sensitivity.

Significance:  Specificity and sensitivity of the iQ-Check STEC method are equivalent to that of the reference method.  It is a reliable and easy-to-use alternative with a reduced time-to-result.