TY - JOUR T1 - Guidelines for optimisation of a multiplex oligonucleotide ligation-PCR for characterisation of microbial pathogens in a microsphere suspension array. JF - Biomed Res Int Y1 - 2015 A1 - Wuyts, VĂ©ronique A1 - Nancy Roosens A1 - Sophie Bertrand A1 - Marchal, Kathleen A1 - Sigrid C.J. De Keersmaecker KW - DNA Primers KW - DNA, Bacterial KW - Humans KW - Microspheres KW - Multiplex Polymerase Chain Reaction KW - Oligonucleotide Probes KW - Salmonella typhimurium AB -

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.

VL - 2015 U1 - http://www.ncbi.nlm.nih.gov/pubmed/25705689?dopt=Abstract M3 - 10.1155/2015/790170 ER -