Development of a genomic DNA reference material for Salmonella enteritidis detection using polymerase chain reaction |
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| Affiliation: | 1. Laboratory of Hematology, University Hospital Saint-Louis, AP-HP, Paris, France;2. EA3518, University Paris Diderot, Paris, France;3. Imagene, Evry, France;4. Laboratory of Hematology, CHRU, Lille, France;5. Laboratory of Hematology, University Hospital, Bordeaux, France;6. Laboratory of Hematology, University Hospital, Toulouse, France;7. Laboratory of Hematology, University Hospital Necker-Enfants-Malades, AP-HP, Paris, France;8. Laboratory of Hematology, University Hospital, Lyon, France;9. Laboratoire de Biopathologie, Institut Paoli Calmettes, Marseille, France;10. Group of Molecular Biologists for Malignant Hemopathies, Toulouse, France;1. Institute for Infectious Diseases, University of Bern, Bern, Switzerland;2. Research Service, Louis Stokes Cleveland Department of Veteran Affairs Medical Center, Cleveland, USA;3. Department of Medicine, Case Western Reserve University, Cleveland, USA;4. Department of Infectious Diseases, Bern University Hospital and University of Bern, Bern, Switzerland;5. Department of Pharmacology, Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, USA |
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| Abstract: | Several rapid methods based on nucleic acids can detect foodborne pathogens, such as Salmonella spp. However, a common reference that enables metrological traceability among measurement results is not available. Reference materials (RM) are thus key to guarantee methodological comparability. This study developed a candidate genomic DNA reference material for Salmonella enteritidis quantification to establish performance conditions and reference values for normalized RM production. The growth of Salmonella enteritidis ATCC® 13076 in Rappaport Vassiliadis selective medium was characterized, and we optimized a method of DNA extraction using cetrimonium bromide (CTAB) and LiCl.In a first stage six concentrations of DNA were prepared with and without yeast RNA (40 ng/μL) to evaluate its effect as a stabilizer in terms of homogeneity and short-term stability. Based on the findings, in a second stage two DNA concentrations were prepared and a reference value with its uncertainty was assigned based on the results of characterization, homogeneity, and stability studies using digital polymerase chain reaction and the gene targets, invA, ttr, and hilA. The material was stable for 9 months at 4 °C, with a expanded uncertainty contribution range of 11%–14%. The novel candidate RM is the first to be developed nationwide and will improve the quality of measurements in the area of food safety. |
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| Keywords: | Droplet digital PCR Salmonella spp. Standard reference material Bioanalysis Food safety |
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