Applicability of the QIAxcel System and a Multiplex PCR for Shiga Toxin-producing Escherichia coli Detection

Introduction: The major STEC serogroups that cause human infections are O157, O26, O103, O111, O121, O45, and O145 (top-7). Four virulence factors: stx1, stx2, eae, ehxA are commonly associated with more severe forms of human infections. The organisms are reside and propagated in the gastrointestinal tract in cattle and are shed in feces, serving as a major source of food and water contamination. An 11-gene multiplex PCR was established to detect the O-antigens of the “top-7” STECs and the 4 virulence factors with manual data interpretation. 

Purpose: To evaluate automatic data interpretations by the Peak-Calling function in the QIAxcel software, and compare with manual interpretation results on amplicons generated by the 11-gene multiplex PCR.

Methods: A total of 185 enriched cattle fecal swab pools (5 per pool) were amplified for 11-genes described above. PCR products were directly run on the QIAxcel without further manipulation. Positive amplifications of each of the 11 genes for each sample were manually interpreted. The results were then compared with data generated automatically by the Peak-Calling method.

Results: Most samples (86.5%) were positive for at least one of the seven O-types, and two virulence genes. Positive bands for individual sample ranged from 2-9 (some samples were positive for more than one O-types), with average of 5 bands.  The total number of bands in these 185 samples was 931. Samples were 61.6%, 28.1% and 19.5% positive for O157, O26 and O103 E. coli serogroups, respectively. Bands that manually identified were 98.9% the same as those identified by Peak-Calling interpretations.

Significance: From the 185 samples tested, Peak-Calling method identified the same numbers and same sizes of bands to that generated by manual interpretations.  Applying the Peak-Calling method post an 11-gene multiplex PCR will significantly save personnel time. The method will also reduce potential interpretation errors generated by manual interpretations.