Purpose: The current study compares microfluidic separation by the Agilent 2100 Bioanalyzer to gel electrophoresis as an alternative for vibrio confirmation.
Methods: DNA templates were prepared from overnight broth cultures as described in the BAM. The organisms tested were previously characterized for the species specific and virulence markers listed below. PCR was performed as described in the BAM for V. cholerae cholera toxin (777 bp fragment of ctxAB), V. vulnificus species (519 bp fragment of vvh) and V. parahaemolyticus (triplex assay for 450 bp fragment of tlh species specific marker, 500 bp fragment of trh virulence marker and 270 bp fragment of tdh virulence marker). Product from each PCR was visualized by gel electrophoresis as described in the BAM and by microfluidic separation on “DNA chips” with the Agilent 2100.
Results: Of 51 V. cholerae isolates tested, ctx was detected after PCR in the same 16 isolates by both gel electrophoresis and by the Agilent. The vvh gene was detected in 52/52 isolates by gel electrophoresis and in all 53 V. vulnificus isolates with microfluidic separation. The V. parahaemolyticus species specific marker, tlh, was detected in 52/53 isolates by Agilent and gel electrophoresis. The trh marker was identified in 26/53 V. parahaemolyticus isolates with the Agilent and in 25/53 with gel electrophoresis. The tdh gene was detected in 41/53 V. parahaemolyticus isolates by the microfluidic separation technique and 40/53 by gel electrophoresis.
Significance: These results indicate that microfluidic separation is a suitable alternative to gel electrophoresis for sizing and visualization of PCR fragments for the identification and characterization of pathogenic Vibrio spp.