Abstract
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.
Highlights
Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a rapid method for simultaneous detection of multiple molecular markers within a single reaction
Compared to other ligation-based methodologies such as multiplex ligation-dependent probe amplification (MLPA) or the oligonucleotide ligation assay (OLA), where multiplexed PCR is the limiting factor of high multiplexing, in MOL-PCR the ligation step represents the actual detection event and the subsequent PCR does not serve for amplification of template DNA10
No differences were observed in the MOL-PCR optimization analyses when utilizing genomic DNA (gDNA) templates isolated by two distinct methods
Summary
Multiplex oligonucleotide ligation-PCR (MOL-PCR) is a rapid method for simultaneous detection of multiple molecular markers within a single reaction. The multiplex oligonucleotide ligation-PCR (MOL-PCR) first described by Deshpande et al.[4] combines analysis of multiple types of molecular markers in a single multiplex reaction[4,6,7] and is capable of specific sequence detection in parallel with SNP identification or detection of indels (insertion/deletion) Owing to these features, MOL-PCR represents a sensitive tool for complex screening of pathogens (virus, bacteria, fungi) in various matrices or to establish the presence of genes responsible for antibiotic resistance. Carrying out of a typical MOL-PCR assay involves three crucial steps: multiplex oligonucleotide ligation of specific probes for detection of molecular markers, PCR for signal amplification, and the hybridization of PCR products to magnetic microspheres followed by signal detection on MAGPIX (Fig. 1).
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