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.