Abstract
BackgroundFast molecular detection methods benefit from ready-to-run lab-on-a-chip molecular assays with minimum preparation time. Detection efficiency of such methods can improve if multiple targets are detected simultaneously per given reaction. Detection of food pathogens, i.e. Escherichia coli (E. coli), is generally performed in two stages with the detection of multiple targets in each stage.With simultaneous testing, screening for pathogens is fast and efficient.ResultsIn this study, we show the application of multiplex PCR performed on a ready-made cassette to detect 10 targets each for eight samples known to harbor E. coli. In cassette PCR, the aluminum cassette (38.6 mm × 31.4 mm) contains 10 trenches having a total of 50 capillaries with microliter volumes of desiccated acrylamide gels holding all reagents required for the PCR including internal positive and negative controls. The gel contains LCGreen dye to detect double stranded DNA. Fluorescence monitoring allows the detection of the amplified products by melt curve analysis. In this application, each of the five capillaries in a given trench contains two of the primer sets for the detection of 10 targets in pathogenic E. coli, namely, O157, Eae, Stx1, Stx2 and six O-antigen genes. Primer specificity was confirmed. Each trench tests one sample. Eight minimally processed enriched beef carcass swab samples were analyzed for parallel detection of 10 targets within 1 h and 15 min. Samples were delivered to the capillaries by capillary forces thereby hydrating the gels. Multiplex cassette PCR results were confirmed with conventional multiplex PCRs performed in a commercial real-time PCR system.ConclusionsCassette PCR technology is ideally suited to multi-target detection of pathogens in food products. The cassette performs multiple PCR reactions in parallel, with multiplex detection of targets within each reaction unit. Cassette PCR/ melt curve analysis results for the simultaneous detection of 10 targets of pathogenic E.coli in beef carcass swab samples were confirmed with a conventional real-time PCR/ melt curve analysis as well as with agarose gel electrophoresis. Although designed for the detection of E. coli, this multiplex cassette PCR technique can be applied to any other assay where the fast detection of multiple targets is required.
Highlights
Fast molecular detection methods benefit from ready-to-run lab-on-a-chip molecular assays with minimum preparation time
We demonstrate the ability of cassette Polymerase Chain Reaction (PCR) to carry out multiplex detection of markers for enterohemorrhagic Escherichia coli in enriched beef carcass swab samples
Eight enriched and minimally processed beef carcass swab samples were amplified by PCR and the products were melted in the first 8 trenches while the negative and positive controls were amplified and melted in the 9th and 10th trenches respectively
Summary
Fast molecular detection methods benefit from ready-to-run lab-on-a-chip molecular assays with minimum preparation time. Examples where multiplex PCRs can be Multiplex real-time PCRs are mostly performed with many primers coupled with probes that are labeled with different colored fluorophores [11] The detection of such PCR products requires a sophisticated instrument with a wide spectrum light source to excite each fluorophore as well as multiple light filters to measure the fluorescence emission from each dye. Probes labeled with different colored fluorophores as well as the instruments capable of detecting multiple colors are costly Another option would be to do multiplex real-time regular PCRs with primers that amplify products of different sizes that can be resolved with no need for probes, by adding an intercalating fluorescent dye to the reaction, and using the position of the melt peaks to detect amplification of multiple DNA targets [12, 13]. With a single color, since the position of the melt peak of the amplicon is used for the diagnosis, primers must be designed to produce PCR products with distinct melt peaks
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