Advanced search
1 file | 725.82 KB

Guidelines for optimisation of a multiplex oligonucleotide ligation-PCR for characterisation of microbial pathogens in a microsphere suspension array

Author
Organization
Project
Bioinformatics: from nucleotids to networks (N2N)
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.
Keywords
IDENTIFICATION, DEPENDENT PROBE AMPLIFICATION, ASSAY, MLPA, DIAGNOSIS, IBCN, COMBINATION

Downloads

  • BMRI2015-790170.pdf
    • full text
    • |
    • open access
    • |
    • PDF
    • |
    • 725.82 KB

Citation

Please use this url to cite or link to this publication:

Chicago
Wuyts, Véronique, Nancy HC Roosens, Sophie Bertrand, Kathleen Marchal, and Sigrid CJ De Keersmaecker. 2015. “Guidelines for Optimisation of a Multiplex Oligonucleotide ligation-PCR for Characterisation of Microbial Pathogens in a Microsphere Suspension Array.” Biomed Research International.
APA
Wuyts, V., Roosens, N. H., Bertrand, S., Marchal, K., & De Keersmaecker, S. C. (2015). Guidelines for optimisation of a multiplex oligonucleotide ligation-PCR for characterisation of microbial pathogens in a microsphere suspension array. BIOMED RESEARCH INTERNATIONAL.
Vancouver
1.
Wuyts V, Roosens NH, Bertrand S, Marchal K, De Keersmaecker SC. Guidelines for optimisation of a multiplex oligonucleotide ligation-PCR for characterisation of microbial pathogens in a microsphere suspension array. BIOMED RESEARCH INTERNATIONAL. 2015;
MLA
Wuyts, Véronique, Nancy HC Roosens, Sophie Bertrand, et al. “Guidelines for Optimisation of a Multiplex Oligonucleotide ligation-PCR for Characterisation of Microbial Pathogens in a Microsphere Suspension Array.” BIOMED RESEARCH INTERNATIONAL (2015): n. pag. Print.
@article{5945236,
  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.},
  articleno    = {790170},
  author       = {Wuyts, V{\'e}ronique and Roosens, Nancy HC and Bertrand, Sophie and Marchal, Kathleen and De Keersmaecker, Sigrid CJ},
  issn         = {2314-6133},
  journal      = {BIOMED RESEARCH INTERNATIONAL},
  keyword      = {IDENTIFICATION,DEPENDENT PROBE AMPLIFICATION,ASSAY,MLPA,DIAGNOSIS,IBCN,COMBINATION},
  language     = {eng},
  pages        = {10},
  title        = {Guidelines for optimisation of a multiplex oligonucleotide ligation-PCR for characterisation of microbial pathogens in a microsphere suspension array},
  url          = {http://dx.doi.org/10.1155/2015/790170},
  year         = {2015},
}

Altmetric
View in Altmetric
Web of Science
Times cited: