A multiplex oligonucleotide ligation-PCR as a complementary tool for subtyping of Salmonella Typhimurium

Wuyts, Véronique; Mattheus, Wesley; Roosens, Nancy; Marchal, Kathleen; Bertrand, Sophie; Keersmaecker, Sigrid C. J. De

doi:10.1007/s00253-015-6831-7

Cited by 12 publications

(16 citation statements)

References 64 publications

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“…1,4,[ 5 ],12:i:- and S . Typhimurium isolates with frequently occurring combinations of MOL-PCR profiles (15-1-1 and 15-3-1), phage types (DT193 and DT120) and MLVA profiles (3-12-10-NA-211 and 3-13-11-NA-211) [ 41 ]. Phage type and MLVA data were provided by the NRCSS and were collected as described by Wuyts et al [ 42 ].…”

Section: Methodsmentioning

confidence: 99%

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Comparison of SNP-based subtyping workflows for bacterial isolates using WGS data, applied to Salmonella enterica serotype Typhimurium and serotype 1,4,[5],12:i:-

et al. 2018

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Whole genome sequencing represents a promising new technology for subtyping of bacterial pathogens. Besides the technological advances which have pushed the approach forward, the last years have been marked by considerable evolution of the whole genome sequencing data analysis methods. Prior to application of the technology as a routine epidemiological typing tool, however, reliable and efficient data analysis strategies need to be identified among the wide variety of the emerged methodologies. In this work, we have compared three existing SNP-based subtyping workflows using a benchmark dataset of 32 Salmonella enterica subsp. enterica serovar Typhimurium and serovar 1,4,[5],12:i:- isolates including five isolates from a confirmed outbreak and three isolates obtained from the same patient at different time points. The analysis was carried out using the original (high-coverage) and a down-sampled (low-coverage) datasets and two different reference genomes. All three tested workflows, namely CSI Phylogeny-based workflow, CFSAN-based workflow and PHEnix-based workflow, were able to correctly group the confirmed outbreak isolates and isolates from the same patient with all combinations of reference genomes and datasets. However, the workflows differed strongly with respect to the SNP distances between isolates and sensitivity towards sequencing coverage, which could be linked to the specific data analysis strategies used therein. To demonstrate the effect of particular data analysis steps, several modifications of the existing workflows were also tested. This allowed us to propose data analysis schemes most suitable for routine SNP-based subtyping applied to S. Typhimurium and S. 1,4,[5],12:i:-. Results presented in this study illustrate the importance of using correct data analysis strategies and to define benchmark and fine-tune parameters applied within routine data analysis pipelines to obtain optimal results.

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Section: Methodsmentioning

confidence: 99%

“…Phage type and MLVA data were provided by the NRCSS and were collected as described by Wuyts et al [ 42 ]. Multiplex Oligonucleotide Ligation-PCR (MOL-PCR) data has been determined previously by Wuyts et al [ 41 ]. Sequence type was determined from the WGS data using MLST-1.8 Server from Centre of Genomic Epidemiology [ 43 ].…”

Section: Methodsmentioning

confidence: 99%

Comparison of SNP-based subtyping workflows for bacterial isolates using WGS data, applied to Salmonella enterica serotype Typhimurium and serotype 1,4,[5],12:i:-

et al. 2018

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“…In the authors' MOL-PCR for characterization of Salmonella enterica subsp. enterica serovar Typhimurium (Wuyts et al, 2015a;Wuyts et al, 2015b), the target-specific sequences of upstream and downstream probes ranges from 16 to 35 bp.…”

Section: Methodsmentioning

confidence: 99%

“…MOL-PCR can be used to detect different types of molecular markers, such as unique sequences, singlenucleotide polymorphisms (SNPs) and indels in a single multiplex reaction . As the MOL-PCR procedure makes use of the Luminex xTAG technology, offering 150 different precoupled microspheres, maximum 150 distinct molecular markers can be assessed in 1 assay, but typical MOL-PCR procedures for characterization of bacteria use around 13-plex up to 22-plex assays Stucki et al, 2012;Thierry et al, 2013;Wuyts et al, 2015a;Wuyts et al, 2015b). In the described MOL-PCR protocol, summarized schematically in Figure 13.15.1, targeted molecular markers present in the isolated genomic DNA are detected in a multiplex oligonucleotide ligation reaction, during which two adjacent probes for each marker are ligated.…”

Section: Introductionmentioning

confidence: 99%

Optimized MOL‐PCR for Characterization of Microbial Pathogens

et al. 2016

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Characterization of microbial pathogens is necessary for surveillance, outbreak detection, and tracing of outbreak sources. This unit describes a multiplex oligonucleotide ligation‐PCR (MOL‐PCR) optimized for characterization of microbial pathogens. With MOL‐PCR, different types of markers, like unique sequences, single‐nucleotide polymorphisms (SNPs) and indels, can be simultaneously analyzed in one assay. This assay consists of a multiplex ligation for detection of the markers, a singleplex PCR for signal amplification, and hybridization to MagPlex‐TAG beads for readout on a Luminex platform after fluorescent staining. The current protocol describes the MOL‐PCR, as well as methods for DNA isolation, probe design, and data interpretation and it is based on an optimized MOL‐PCR assay for subtyping of Salmonella Typhimurium. © 2016 by John Wiley & Sons, Inc.

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“…Numerous studies have been conducted to detect and characterize Salmonella in poultry, poultry products, and feeds using PCR assays to target selected antibiotic resistance or virulence genes along with genus-, species-, and serotype-speciic genes [16,[37][38][39][40].…”

Section: Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

Current and Emerging Innovations for Detection of Food-Borne Salmonella

Wu¹,

Zeng²

2017

Current Topics in Salmonella and Salmonellosis

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Salmonella is one of the leading causes of food-borne illnesses worldwide, and one of the main contributors to salmonellosis is the consumption of contaminated egg, poultry, pork, beef, and milk products. Since deleterious efects of Salmonella on public health and the economy continue to occur, improving safety of food products by early detection of food-borne pathogens would be considered an important component for limiting exposure to Salmonella contamination. Therefore, there is an ongoing need to develop more advanced detection methods that can identify Salmonella accurately and rapidly in foods before they reach consumers. In the past three decades, there have been increasing eforts toward developing and improving rapid pathogen detection and characterization methodologies for application to food products. In this chapter, we discuss molecular methods for detection, identiication, and genetic characterization of Salmonella in food. In addition, the advantages and disadvantages of the established and emerging rapid detection methods are addressed here. The methods with potential application to the industry are highlighted in this chapter.

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A multiplex oligonucleotide ligation-PCR as a complementary tool for subtyping of Salmonella Typhimurium

Cited by 12 publications

References 64 publications

Comparison of SNP-based subtyping workflows for bacterial isolates using WGS data, applied to Salmonella enterica serotype Typhimurium and serotype 1,4,[5],12:i:-

Comparison of SNP-based subtyping workflows for bacterial isolates using WGS data, applied to Salmonella enterica serotype Typhimurium and serotype 1,4,[5],12:i:-

Optimized MOL‐PCR for Characterization of Microbial Pathogens

Current and Emerging Innovations for Detection of Food-Borne Salmonella

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