High-Throughput Detection of Shiga Toxin-Producing E. coli Using Multiplex PCR and the QIAxcel System

Shiga toxin-producing E. coli (STEC) causes approximately 176,000 human illnesses in the US each year. These organisms reside and propagate in the gastrointestinal tract of cattle and serve as a major source of food and water contamination when they are shed in the feces. The "top 7" STEC serogroups causing human infections are O157, O26, O103, O111, O121, O45, and O145. Four major virulence factors are commonly associated with more severe infections: Shiga toxin 1 and 2 (stx1, stx2), intimin (eae), and enterohemolysin (ehxA).

We have developed an 11-gene multiplex endpoint PCR assay to detect and differentiate the "top 7" STEC serogroups and the 4 major virulence factors in a high-throughput manner. Most multiplex real-time PCR assays are limited to 2-3 targets due to design difficulties. End-point PCR can detect more targets than real-time PCR, but is laborious due to the need to visualize results using agarose gels. We have improved the procedure by replacing agarose gel analysis with the QIAxcel automated, high-throughput capillary electrophoresis system. The peak-calling function in the QIAxcel ScreenGel software automatically interprets the presence or absence of each band in every sample, further improving the manual process to save personnel time and reduce potential for human error in manual interpretations.

Dr. Jianfa Bai

Dr. Jianfa Bai is an assistant professor and molecular diagnostician in the Kansas State Veterinary Diagnostic Laboratory (KSVDL), Kansas State University. He leads the effort of new assay development at KSVDL and has developed a few dozen new molecular detection assays together with his team. He is also one of the lead persons developing methods for the detection and differentiation of Shiga toxin-producing Escherichia coli strains. Dr. Bai is experienced in microbiology, molecular biology and basic bioinformatics. He established and served as the director of the KSU Gene Expression Facility (now the Integrated Genomics Facility) prior to his current position.