title: On-chip parallel detection of foodborne pathogens using loop-mediated isothermal amplification.
abstract: 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.
authors.affiliation: Department of Electrical and Computer Engineering, William L. Everitt Laboratory, University of Illinois at Urbana-Champaign, 1406 W. Green St., Urbana, IL 61801, USA.
authors.forename: Carlos
authors.initials: C
authors.lastname: Duarte
authors.forename: Eric
authors.initials: E
authors.lastname: Salm
authors.forename: Brian
authors.initials: B
authors.lastname: Dorvel
authors.forename: Bobby
authors.initials: B
authors.lastname: Reddy
authors.forename: Rashid
authors.initials: R
authors.lastname: Bashir
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000302:isolation & purification; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology / Q000379:methods*; D008089:Listeria monocytogenes / Q000302:isolation & purification; D008845:Microinjections; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012475:Salmonella / Q000302:isolation & purification; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial