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Molecular Subtyping of Salmonella Typhimurium with Multiplex Oligonucleotide Ligation-PCR (MOL-PCR)

  • Véronique Wuyts
  • Wesley Mattheus
  • Nancy H. C. Roosens
  • Kathleen Marchal
  • Sophie Bertrand
  • Sigrid C. J. De Keersmaecker
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1616)

Abstract

A multiplex oligonucleotide ligation-PCR (MOL-PCR) assay is a valuable high-throughput technique for the detection of bacteria and viruses, for characterization of pathogens and for diagnosis of genetic diseases, as it allows one to combine different types of molecular markers in a high-throughput multiplex assay. A MOL-PCR assay starts with a multiplex oligonucleotide ligation reaction for detection of the molecular marker, followed by a singleplex PCR for signal amplification and analysis of the MOL-PCR products on a Luminex platform. This last step occurs through a liquid bead suspension array in which the MOL-PCR products are hybridized to MagPlex-TAG beads.

In this chapter, we describe the complete procedure for a MOL-PCR assay for subtyping of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant S. 1,4[5],12:i:- from DNA isolation through heat lysis up to data interpretation through a Gödel Prime Product. The subtyping assay consists of 50 discriminative molecular markers and two internal positive control markers divided over three MOL-PCR assays.

Key words

MOL-PCR Salmonella Typhimurium Subtyping Luminex Bead suspension array Gödel Prime Product High-throughput assay 

Notes

Acknowledgment

This work was supported by grant P4044.0103 (SalMolType) from the Scientific Institute of Public Health (WIV-ISP – RP/PJ). The National Reference Centre for Salmonella and Shigella is partially supported by the Belgian Ministry of Social Affairs through a fund within the Health Insurance System.

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Véronique Wuyts
    • 1
  • Wesley Mattheus
    • 2
  • Nancy H. C. Roosens
    • 1
  • Kathleen Marchal
    • 3
    • 4
  • Sophie Bertrand
    • 2
  • Sigrid C. J. De Keersmaecker
    • 1
  1. 1.Platform Biotechnology and Molecular BiologyScientific Institute of Public Health (WIV-ISP)BrusselsBelgium
  2. 2.National Reference Centre for Salmonella and Shigella, Bacterial Diseases Division, Communicable and Infectious DiseasesScientific Institute of Public Health (WIV-ISP)BrusselsBelgium
  3. 3.Department of Plant Biotechnology and BioinformaticsGhent UniversityGhentBelgium
  4. 4.Department of Information TechnologyGhent University, IMindsGhentBelgium

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