Issue 7, 2014

Real-time PCR based detection of a panel of food-borne pathogens on a centrifugal microfluidic “LabDisk” with on-disk quality controls and standards for quantification

Abstract

We present an implementation of parallel, real-time PCR based detection of up to 6 different food-borne pathogens on our centrifugal microfluidic “LabDisk” platform. It has the following two novelties: (1) a microfluidic network for integration of positive controls (PCs), no-template controls (NTCs), and standards (STDs) into a centrifugal microfluidic PCR cartridge; (2) a microfluidic unit operation for sequential aliquoting of two liquids of highly different wetting characteristics into fourteen aliquots with 5.8 μL ± 0.3 μL (PCR mastermix) and 6.1 ± 0.8 μL (elution buffer), respectively. The presented “LabDisk” implementation can be used either in a qualitative or quantitative operation mode depending on the prestorage scheme of reagents. In qualitative mode, two DNA samples can be tested per cartridge for the presence of 6 food pathogens (Listeria monocytogenes, Salmonella typhimurium, EHEC, Staphylococcus aureus, Citrobacter freundii and Campylobacter jejuni), including PCs and NTCs. This was proofed for DNA concentrations of 10 pg, 1 pg, and 0.1 pg per pathogen. In quantitative mode, one DNA sample per cartridge can be analysed quantitatively for the presence of two pathogens by prestored and on-disk generated standard curves. 50 pg and 500 pg L. monocytogenes genomic DNA samples have been quantified to 83 ± 17 pg and 540 ± 116 pg DNA, respectively, while 50 pg and 500 pg S. typhimurium DNA samples have been quantified to 48 ± 4 pg and 643 ± 211 pg DNA. In both operation modes, the microfluidic routing of the liquids was done by spinning the cartridge on a low-cost centrifugal test rig. For real-time PCR amplification, the cartridge was then transferred into a commercially available thermocycler. The nucleic acid amplification and detection as presented here is fully compatible with upstream DNA extraction as presented previously (Strohmeier et al., Lab Chip, 2013, 13, 146-155). Concatenation of both fluidic structures will enable fully integrated sample-to-answer testing of food-borne pathogens in the future.

Graphical abstract: Real-time PCR based detection of a panel of food-borne pathogens on a centrifugal microfluidic “LabDisk” with on-disk quality controls and standards for quantification

Supplementary files

Article information

Article type
Paper
Submitted
17 Oct 2013
Accepted
26 Dec 2013
First published
06 Jan 2014

Anal. Methods, 2014,6, 2038-2046

Real-time PCR based detection of a panel of food-borne pathogens on a centrifugal microfluidic “LabDisk” with on-disk quality controls and standards for quantification

O. Strohmeier, N. Marquart, D. Mark, G. Roth, R. Zengerle and F. von Stetten, Anal. Methods, 2014, 6, 2038 DOI: 10.1039/C3AY41822G

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