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Optimized MOL-PCR for Characterization of Microbial Pathogens.

Véronique Wuyts^1,2,3, Nancy H C Roosens³, Sophie Bertrand⁴

¹Department of Microbial and Molecular Systems, Centre of Microbial and Plant Genetics, KU Leuven, Leuven, Belgium.

Current Protocols in Cytometry

|January 9, 2016

Summary

This study presents a multiplex oligonucleotide ligation-PCR (MOL-PCR) assay for simultaneous characterization of microbial pathogens. This method enhances pathogen surveillance and outbreak source tracing capabilities.

Keywords:

Luminex MOL-PCR microsphere suspension array xTAG technology

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Area of Science:

Microbiology
Molecular Biology
Genetics

Background:

Accurate characterization of microbial pathogens is crucial for effective disease surveillance, outbreak detection, and source tracing.
Existing methods may lack the capacity for simultaneous analysis of multiple genetic markers.

Purpose of the Study:

To describe an optimized multiplex oligonucleotide ligation-PCR (MOL-PCR) assay for comprehensive microbial pathogen characterization.
To enable simultaneous analysis of diverse genetic markers including unique sequences, single-nucleotide polymorphisms (SNPs), and indels.

Main Methods:

The assay involves multiplex ligation for marker detection, singleplex PCR for signal amplification, and Luminex platform readout using MagPlex-TAG beads.
The protocol details DNA isolation, probe design, and data interpretation, optimized for Salmonella Typhimurium subtyping.

Main Results:

The described MOL-PCR assay allows for simultaneous analysis of multiple genetic markers in a single reaction.
The method provides a robust platform for detailed microbial subtyping and characterization.

Conclusions:

Multiplex oligonucleotide ligation-PCR (MOL-PCR) offers an efficient and versatile tool for microbial pathogen characterization.
This optimized assay improves capabilities for public health surveillance and infectious disease outbreak investigations.