|Duarte-guevara, Paula - Purdue University|
|Duarte-guevara, Carlos - Purdue University|
|Ornob, Akid - Purdue University|
|Bashir, Rashid - Purdue University|
Submitted to: Microfluidics and Nanofluidics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/12/2016
Publication Date: 7/19/2016
Citation: Duarte-Guevara, P., Duarte-Guevara, C., Ornob, A., Bashir, R. 2016. On-chip PMA labeling of foodborne pathogenic bacteria for viable qPCR and qLAMP detection. Microfluidics and Nanofluidics. doi: 10.1007/s104016-1778-2.
Interpretive Summary: The World Health Organization (WHO) established foodborne diseases as one of the eleven priorities and challenges for the millennium. One of the reasons for the persistence and prevalence of foodborne illnesses is the poor performance of current microorganism detection techniques that undermines effective prevention. Many rapid techniques are unable to differentiate between live and dead organisms, and it is the live organisms that are most concerning for food safety. There is a type of molecule, called propidium monoazide (PMA) that can be used to differentiate between live and dead bacteria in standard detection methods based on DNA amplification. In this study, we combined the use of PMA with their developed microfluidic detection technology and were able to specifically and quickly detect harmful bacteria, including E. coli O157 and Salmonella Typhimurium. Because of the small size of the device, the assays are less expensive than many existing technologies. The use of the developed assays will allow the rapid on-site screening tests for viable organisms in food samples, prevent the distribution of contaminated foods, and improve the overall safety of our food supply.
Technical 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 uL/min for complete labeling with the current design.