Novel AlphaPlex Design Enables Rapid Differentiation of Campylobacter Species

Campylobacter jejuni and Campylobacter coli are major foodborne pathogens whose accurate species-level discrimination is important for outbreak investigations as well as rapidly assessing putative antimicrobial resistance profiles and the pathogenic potential of the bacterium. To facilitate species differentiation, a novel assay that integrates the nucleic acid-sensing capability of the oligo-Alpha with the multiplexing capacity of the AlphaPlex bead chemistries was developed. This wash-free system (designated as oligo-Plex) enables the detection and differentiation of C. jejuni and C. coli within a single reaction and can be completed in approximately 75 min. It works by using custom oligonucleotides modified for bead attachment, which hybridize sequentially along Campylobacter's glyA gene and ultimately bridge the donor and acceptor beads. Improvements in assay stringency were made by increasing incubation temperatures, thus allowing the resolution of target from non-target. Comparisons of FITC-europium and DIG-terbium labeling systems revealed superior performance by the FITC-europium pair and suggested that helical positioning and steric accessibility likely influence donor-acceptor efficiency. Maximized signal separation was seen when using terbium for the detection of C. coli and europium for the detection of C. jejuni. Testing was performed in a Tris-based buffer and milk to confirm matrix tolerance, with potential areas for further optimization identified. The oligo-Plex presented here establishes a streamlined, adaptable platform suitable for high-throughput screening of multiple nucleic acid analytes that is readily extendable to a diverse array of pathogens through appropriate oligo selection.