Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a rapid method for simultaneous detection of multiple molecular markers within a single reaction. MOL-PCR is increasingly employed in microbial detection assays, where its ability to facilitate identification and further characterization via simple analysis is of great benefit and significantly simplifies routine diagnostics. When adapted to microsphere suspension arrays on a MAGPIX reader, MOL-PCR has the potential to outperform standard nucleic acid-based diagnostic assays. This study represents the guideline towards in-house MOL-PCR assay optimization using the example of foodborne pathogens (bacteria and parasites) with an emphasis on the appropriate choice of crucial parameters. The optimized protocol focused on specific sequence detection utilizes the fluorescent reporter BODIPY-TMRX and self-coupled magnetic microspheres and allows for a smooth and brisk workflow which should serve as a guide for the development of MOL-PCR assays intended for pathogen detection.

Faculty of Science Department of Botany and Zoology Masaryk University Kotlářská 2 611 37 Brno Czech Republic

Faculty of Science Department of Experimental Biology Masaryk University Kamenice 753 5 625 00 Brno Czech Republic

Veterinary Research Institute Department of Food and Feed Safety Hudcova 296 70 621 00 Brno Czech Republic

Sci Rep. 2019 Apr 4;9(1):5842. doi: 10.1038/s41598-019-42194-x. PubMed

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