title: Optimized MOL-PCR for Characterization of Microbial Pathogens.
abstract: Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation-PCR (MOL-PCR) optimized for characterization of microbial pathogens. With MOL-PCR, different types of markers, like unique sequences, single-nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex-TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL-PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL-PCR assay for subtyping of Salmonella Typhimurium.
authors.affiliation: Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.|Platform Biotechnology and Molecular Biology, Scientific Institute of Public Health (WIV-ISP), Brussels, Belgium.
authors.forename: Véronique
authors.initials: V
authors.lastname: Wuyts
authors.affiliation: Platform Biotechnology and Molecular Biology, Scientific Institute of Public Health (WIV-ISP), Brussels, Belgium.
authors.forename: Nancy H C
authors.initials: NHC
authors.lastname: Roosens
authors.affiliation: National Reference Centre for Salmonella and Shigella, Bacterial Diseases Division, Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP), Brussels, Belgium.
authors.forename: Sophie
authors.initials: S
authors.lastname: Bertrand
authors.affiliation: Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium.|Department of Information Technology, Ghent University, IMinds, Ghent, Belgium.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Marchal
authors.affiliation: Platform Biotechnology and Molecular Biology, Scientific Institute of Public Health (WIV-ISP), Brussels, Belgium.
authors.forename: Sigrid C J
authors.initials: SCJ
authors.lastname: De Keersmaecker
mesh_terms: D001419:Bacteria / Q000145:classification*; D015415:Biomarkers / Q000378:metabolism; D019921:Biotinylation; D004269:DNA, Bacterial / Q000032:analysis; D016521:Electrophoresis, Gel, Pulsed-Field; D005456:Fluorescent Dyes / Q000737:chemistry; D005819:Genetic Markers / Q000235:genetics; D006801:Humans; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry; D010799:Phycoerythrin / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D012486:Salmonella typhimurium / Q000145:classification; D019809:Streptavidin / Q000737:chemistry; D013696:Temperature
chemical_list: D015415:Biomarkers; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D005819:Genetic Markers; D015345:Oligonucleotide Probes; D010799:Phycoerythrin; D019809:Streptavidin
keywords: Luminex; MOL-PCR; microsphere suspension array; xTAG technology