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      Rapid detection and differentiation of Salmonella species, Salmonella Typhimurium and Salmonella Enteritidis by multiplex quantitative PCR

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          Abstract

          A multiplex quantitative PCR (qPCR) was developed and evaluated for the simultaneous detection of Salmonella spp., S. enterica serovar Typhimurium and S. enterica serovar Enteritidis in various (food) matrices. Early and fast detection of these pathogens facilitates effective intervention and prevents further distribution of contaminated food products on the market. Three primer and probe sets were designed to target the invA gene, the STM4200 gene, and the SEN1392 gene to detect and differentiate Salmonella spp., S. Typhimurium, and S. Enteritidis, respectively. The multiplex qPCR targeting these three genes was optimized for efficiency and linearity. By testing 225 Salmonella isolates and 34 non- Salmonella isolates from various sources the inclusivity and exclusivity were determined. The inclusivity of the multiplex qPCR was 100% for all Salmonella isolates, including 72 S. Typhimurium isolates, and 53 S. Enteritidis isolates. The exclusivity for Salmonella spp., S. Typhimurium, and S. Enteritidis was 100%, 94.6%, and 100%, respectively. No positive results were reported for non- Salmonella isolates. The limit of detection (LOD) for the qPCR was determined for the matrices poultry, minced meat, egg, herbs/spices, powdered milk, fish, animal feed, boot-socks with chicken feces and chicken down. LOD values for qPCR and the conventional culture methods were similar, except for the matrix boot-socks and down, for which the LOD for the conventional culture methods performed better than the qPCR method. In conclusion, the multiplex qPCR assay developed allows for rapid screening of Salmonella spp., S. Typhimurium, and S. Enteritidis in various (food) matrices.

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          Most cited references22

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          The global burden of nontyphoidal Salmonella gastroenteritis.

          To estimate the global burden of nontyphoidal Salmonella gastroenteritis, we synthesized existing data from laboratory-based surveillance and special studies, with a hierarchical preference to (1) prospective population-based studies, (2) "multiplier studies," (3) disease notifications, (4) returning traveler data, and (5) extrapolation. We applied incidence estimates to population projections for the 21 Global Burden of Disease regions to calculate regional numbers of cases, which were summed to provide a global number of cases. Uncertainty calculations were performed using Monte Carlo simulation. We estimated that 93.8 million cases (5th to 95th percentile, 61.8-131.6 million) of gastroenteritis due to Salmonella species occur globally each year, with 155,000 deaths (5th to 95th percentile, 39,000-303,000 deaths). Of these, we estimated 80.3 million cases were foodborne. Salmonella infection represents a considerable burden in both developing and developed countries. Efforts to reduce transmission of salmonellae by food and other routes must be implemented on a global scale.
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            Salmonella serotype determination utilizing high-throughput genome sequencing data.

            Serotyping forms the basis of national and international surveillance networks for Salmonella, one of the most prevalent foodborne pathogens worldwide (1-3). Public health microbiology is currently being transformed by whole-genome sequencing (WGS), which opens the door to serotype determination using WGS data. SeqSero (www.denglab.info/SeqSero) is a novel Web-based tool for determining Salmonella serotypes using high-throughput genome sequencing data. SeqSero is based on curated databases of Salmonella serotype determinants (rfb gene cluster, fliC and fljB alleles) and is predicted to determine serotype rapidly and accurately for nearly the full spectrum of Salmonella serotypes (more than 2,300 serotypes), from both raw sequencing reads and genome assemblies. The performance of SeqSero was evaluated by testing (i) raw reads from genomes of 308 Salmonella isolates of known serotype; (ii) raw reads from genomes of 3,306 Salmonella isolates sequenced and made publicly available by GenomeTrakr, a U.S. national monitoring network operated by the Food and Drug Administration; and (iii) 354 other publicly available draft or complete Salmonella genomes. We also demonstrated Salmonella serotype determination from raw sequencing reads of fecal metagenomes from mice orally infected with this pathogen. SeqSero can help to maintain the well-established utility of Salmonella serotyping when integrated into a platform of WGS-based pathogen subtyping and characterization.
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              Diagnostic real-time PCR for detection of Salmonella in food.

              A robust 5' nuclease (TaqMan) real-time PCR was developed and validated in-house for the specific detection of Salmonella in food. The assay used specifically designed primers and a probe target within the ttrRSBCA locus, which is located near the Salmonella pathogenicity island 2 at centisome 30.5. It is required for tetrathionate respiration in Salmonella. The assay correctly identified all 110 Salmonella strains and 87 non-Salmonella strains tested. An internal amplification control, which is coamplified with the same primers as the Salmonella DNA, was also included in the assay. The detection probabilities were 70% when a Salmonella cell suspension containing 10(3) CFU/ml was used as a template in the PCR (5 CFU per reaction) and 100% when a suspension of 10(4) CFU/ml was used. A pre-PCR sample preparation protocol including a preenrichment step in buffered peptone water followed by DNA extraction-purification was applied when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for Salmonella. The diagnostic accuracy was shown to be 100% compared to the traditional culture method. The overall analysis time of the PCR method was approximately 24 h, in contrast to 4 to 5 days of analysis time for the traditional culture method. This methodology can contribute to meeting the increasing demand of quality assurance laboratories for standard diagnostic methods. Studies are planned to assess the interlaboratory performance of this diagnostic PCR method.
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                Author and article information

                Contributors
                Role: ConceptualizationRole: MethodologyRole: ValidationRole: Writing – original draftRole: Writing – review & editing
                Role: Formal analysisRole: Investigation
                Role: InvestigationRole: ValidationRole: Writing – review & editing
                Role: SupervisionRole: ValidationRole: Writing – review & editing
                Role: InvestigationRole: Writing – review & editing
                Role: SupervisionRole: Writing – original draftRole: Writing – review & editing
                Role: ConceptualizationRole: MethodologyRole: SupervisionRole: ValidationRole: VisualizationRole: Writing – original draftRole: Writing – review & editing
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                25 October 2018
                2018
                : 13
                : 10
                : e0206316
                Affiliations
                [001]Netherlands Food and Consumer Product Safety Authority, Directorate Enforcement, Laboratories division, Laboratory for Feed and Food Safety & Product Safety, Wageningen, the Netherlands
                University of Campinas, BRAZIL
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                [¤]

                Current address: Netherlands Food and Consumer Product Safety Authority, Directorate Inspection, Design & Services division, Slaughterhouse team, Utrecht, the Netherlands

                Author information
                http://orcid.org/0000-0002-3432-5626
                http://orcid.org/0000-0003-0947-1258
                Article
                PONE-D-18-21520
                10.1371/journal.pone.0206316
                6201931
                30359449
                03cdd418-6ee6-49d0-b243-292a7306acfa
                © 2018 Heymans et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 20 July 2018
                : 10 October 2018
                Page count
                Figures: 2, Tables: 4, Pages: 15
                Funding
                The authors received no specific funding for this work.
                Categories
                Research Article
                Medicine and Health Sciences
                Infectious Diseases
                Bacterial Diseases
                Salmonella
                Salmonella Typhimurium
                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
                Bacterial Pathogens
                Salmonella
                Salmonella Typhimurium
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Salmonella
                Salmonella Typhimurium
                Biology and Life Sciences
                Organisms
                Bacteria
                Enterobacteriaceae
                Salmonella
                Salmonella Typhimurium
                Biology and Life Sciences
                Agriculture
                Animal Products
                Meat
                Biology and Life Sciences
                Nutrition
                Diet
                Food
                Meat
                Medicine and Health Sciences
                Nutrition
                Diet
                Food
                Meat
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Amniotes
                Birds
                Fowl
                Gamefowl
                Chickens
                Biology and Life Sciences
                Organisms
                Eukaryota
                Animals
                Vertebrates
                Amniotes
                Birds
                Poultry
                Chickens
                Medicine and Health Sciences
                Infectious Diseases
                Bacterial Diseases
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                Biology and Life Sciences
                Microbiology
                Medical Microbiology
                Microbial Pathogens
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                Salmonella
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
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                Infectious Diseases
                Bacterial Diseases
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                Biology and Life Sciences
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                Medical Microbiology
                Microbial Pathogens
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                Salmonella Enterica
                Medicine and Health Sciences
                Pathology and Laboratory Medicine
                Pathogens
                Microbial Pathogens
                Bacterial Pathogens
                Salmonella
                Salmonella Enterica
                Biology and Life Sciences
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                Enterobacteriaceae
                Salmonella
                Salmonella Enterica
                Biology and Life Sciences
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                Molecular Biology Techniques
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                Research and Analysis Methods
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