title: Guidelines for optimisation of a multiplex oligonucleotide ligation-PCR for characterisation of microbial pathogens in a microsphere suspension array.
abstract: With multiplex oligonucleotide ligation-PCR (MOL-PCR) different molecular markers can be simultaneously analysed in a single assay and high levels of multiplexing can be achieved in high-throughput format. As such, MOL-PCR is a convenient solution for microbial detection and identification assays where many markers should be analysed, including for routine further characterisation of an identified microbial pathogenic isolate. For an assay aimed at routine use, optimisation in terms of differentiation between positive and negative results and of cost and effort is indispensable. As MOL-PCR includes a multiplex ligation step, followed by a singleplex PCR and analysis with microspheres on a Luminex device, several parameters are accessible for optimisation. Although MOL-PCR performance may be influenced by the markers used in the assay and the targeted bacterial species, evaluation of the method of DNA isolation, the probe concentration, the amount of microspheres, and the concentration of reporter dye is advisable in the development of any MOL-PCR assay. Therefore, we here describe our observations made during the optimisation of a 20-plex MOL-PCR assay for subtyping of Salmonella Typhimurium with the aim to provide a possible workflow as guidance for the development and optimisation of a MOL-PCR assay for the characterisation of other microbial pathogens.
authors.affiliation: Department of Microbial and Molecular Systems, KU Leuven, Kasteelpark Arenberg 20, Bus 2460, 3001 Leuven, Belgium ; Platform Biotechnology and Molecular Biology, Scientific Institute of Public Health (WIV-ISP), Juliette Wytsmanstraat 14, 1050 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), Juliette Wytsmanstraat 14, 1050 Brussels, Belgium.
authors.forename: Nancy H C
authors.initials: NH
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), Juliette Wytsmanstraat 14, 1050 Brussels, Belgium.
authors.forename: Sophie
authors.identifier: 0000-0002-4170-5168
authors.initials: S
authors.lastname: Bertrand
authors.affiliation: Department of Microbial and Molecular Systems, KU Leuven, Kasteelpark Arenberg 20, Bus 2460, 3001 Leuven, Belgium ; Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052 Ghent, Belgium ; Department of Information Technology, Ghent University, IMinds, Gaston Crommenlaan 8, 9050 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), Juliette Wytsmanstraat 14, 1050 Brussels, Belgium.
authors.forename: Sigrid C J
authors.initials: SC
authors.lastname: De Keersmaecker
mesh_terms: D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008863:Microspheres; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D015345:Oligonucleotide Probes; D012486:Salmonella typhimurium / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D015345:Oligonucleotide Probes