On-chip parallel detection of foodborne pathogens using loop-mediated isothermal amplification

• Published in: Biomedical microdevices Vol. 15; no. 5; pp. 821 - 830
• Main Authors: Duarte, Carlos, Salm, Eric, Dorvel, Brian, Reddy, Bobby, Bashir, Rashid
• Format: Journal Article
• Language: English
• Published: Boston Springer US 01.10.2013; Springer Nature B.V
• Subjects: Biological and Medical Physics; Biomedical Engineering and Bioengineering; Biophysics; Disease prevention; DNA Primers - genetics; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Engineering; Engineering Fluid Dynamics; Escherichia coli; Escherichia coli - isolation & purification; Food contamination & poisoning; Food Contamination - analysis; Food Microbiology - methods; Listeria monocytogenes; Listeria monocytogenes - isolation & purification; Microinjections; Nanotechnology; Nucleic Acid Amplification Techniques - methods; Oligonucleotide Array Sequence Analysis - methods; Pathogens; Salmonella; Salmonella - isolation & purification; Sensitivity and Specificity; Multiplexed screening; Loop-mediated isothermal amplification; Primer dehydration; Miniaturized DNA amplification; Silane passivation
• ISSN: 1387-2176; 1572-8781; 1572-8781
• DOI: 10.1007/s10544-013-9769-5

According to estimates issued by the Center for Disease Control and Prevention, one out of six Americans will get sick during this year due to consumption of contaminated products and there will be 50,000 related hospitalizations. To control and treat the responsible foodborne diseases, rapid and accurate detection of pathogens is extremely important. A portable device capable of performing nucleic acid amplification will enable the effective detection of infectious agents in multiple settings, leading to better enforcement of food safety regulations. This work demonstrates the multiplexed detection of food pathogens through loop-mediated isothermal amplification on a silicon chip. Silane passivation is used to prevent the adsorption of the polymerase on silicon oxide, which can severely inhibit nucleic acid amplification. We demonstrate the multiplexed screening of virulence genes of Listeria monocytogenes , Escherichia coli , and Salmonella by dehydrating the corresponding primers in oxidized silicon wells. Droplets of 30 nL with reagents for nucleic acid amplification and lysate of suspected pathogens are arrayed on micro-machined wells with an automated microinjection system. We show that dehydrated primers re-suspend when other reagents are microinjected, and the resulting mix can be used to specifically amplify the targeted gene. Results of characterization experiments demonstrate sensitivity down to a few templates per reaction, specificity that enables multiplexed screening, and robustness that allows amplification without DNA extraction.