Streamlining a DNA-based Biosensor Detection System for the Detection of Non-PCR Amplified Genomic Pathogenic DNA Targets in Food

Introduction: Our DNA-based biosensor, which utilizes gold nanoparticles (AuNPs) for signal amplification and magnetic nanoparticles (MNPs) for easy and clean separation from samples, has been shown to detect non-PCR amplified genomic DNA targets (DNAt) from bacterial pathogens.  While this detection system can provide detection within an eight-hour window, we hypothesize it could be further streamlined to reduce the overall time to detection, as this would further reduce the overall cost of using this system. 

Purpose: The purpose of this study is to reduce the amount of time it takes go from sample preparation to detection, from seven hours to four hours, without reducing the quality or sensitivity of detection.

Methods: Genomic DNA was extracted from Salmonella Enteritidis and E. coli cultures using either Trizol® or phenol/alcohol method. For extraction, all incubation/centrifugation periods were reduced by 50%, while controls used the standard protocols. For detection, all incubation periods of the biosensor were reduced by 50%, while the controls used the published method. DNA yields were calculated using a nanophotometer. DNAt (50 ng/ml) were detected by hybridizing the DNAt into a sandwich-like structure consisting of MNPs/DNAt/AuNPs, which were then placed onto screen-printed carbon electrodes to detect the voltammetric peaks of gold using differential pulse voltammetry.  All samples were run in triplicate.

Results: DNA yields were similar (P ≥ 0.05) between the normal and reduced incubation/centrifugation protocols.  Equally, reducing the incubation periods of the biosensor system did not impact its ability to detect non-PCR amplified genomic DNAt compared to the established method (P ≥ 0.05); peaks ranging from 2.5 x 10^-5 A to 4.8 x 10^-5 A were achieved using both methods. 

Significance: These findings suggest that reducing the incubation periods within the DNA extraction method and biosensor system did not impact the overall sensitivity of this detection method, and that it can effectively detect pathogenic DNAt within a four-hour window.