On-chip PMA labeling of foodborne pathogenic bacteria for viable qPCR and qLAMP detection

Abstract

Propidium monoazide (PMA) is a membrane impermeable molecule that covalently bonds to double stranded DNA when exposed to light and inhibits the polymerase activity, thus enabling DNA amplification detection protocols that discriminate between viable and non-viable entities. Here, we present a microfluidic device for inexpensive, fast, and simple PMA labeling for viable qPCR and qLAMP assays. The three labeling stages of mixing, incubation, and cross-linking are completed within a microfluidic device that is designed with Tesla structures for passive microfluidic mixing, bubble trappers to improve flow uniformity, and a blue LED to cross-link the molecules. Our results show that the on-chip PMA labeling is equivalent to the standard manual protocols and prevents the replication of DNA from non-viable cells in amplification assays. However, the on-chip process is faster and simpler (30 min of hands-off work), has a reduced likelihood of false negatives, and it is less expensive because it only uses 1/20th of the reagents normally consumed in standard bench protocols. We used our microfluidic device to perform viable qPCR and qLAMP for the detection of S. typhi and E. coli O157. With this device, we are able to specifically detect viable bacteria, with a limit of detection of 7.6 × 103 and 1.1 × 103 CFU/mL for S. typhi and E. coli O157, respectively, while eliminating amplification from non-viable cells. Furthermore, we studied the effects of greater flow rates to expedite the labeling process and identified a maximum flow rate of 0.7 μL/min for complete labeling with the current design.