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      Differentiation of stx1A gene for detection of Escherichia coli serotype O157: H7 and Shigella dysenteriae type 1 in food samples using high resolution melting curve analysis

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          Abstract

          Escherichia coli serotype O157: H7 and Shigella dysenteriae type 1 as the Shiga toxin‐producing bacteria cause some acute gastrointestinal and extraintestinal diseases such as hemorrhagic uremic syndrome and bloody diarrhea in human. Stx genes are the key virulence factors in these pathogens. The aim of this study was to develop HRMA assay to differentiate stx1A gene for detection of E. coli serotype O157: H7 and Sh. dysenteriae type 1 and determine the prevalence of these pathogens in food samples using this method. PCR‐HRMA assay and gold standard methods have been carried out for identification of pathogens among 135 different food samples. We found HRMA method a sensitive and specific assay (100 and 100%, respectively) for differentiation of stx1A gene, consequently, detection of these pathogens in food samples. Also, the highest prevalence of E. coli serotype O157: H7 and Sh. dysenteriae type 1 harboring stx1A gene was observed in raw milk and vegetable salad samples, respectively. HRMA as a rapid, inexpensive, sensitive and specific method is suggested to be used for differentiation of stx1A gene to detect E. coli serotype O157: H7 and Sh. dysenteriae type 1 as the key pathogens for safety evaluation of food samples.

          Abstract

          In this paper, we developed high resolution melting curve analysis method to differentiate stx1A gene to detect Escherichia coli serotype O157: H7 and Shigella dysenteriae strains in food samples. We found this method specific and sensitive for detection of these pathogens in food samples. Also, we investigated the prevalence of these foodborne pathogens in food samples using this method. We observed the highest prevalence of E. coli serotype O157: H7 and Sh. dysenteriae type 1 in raw milk and vegetable salad samples, respectively. We found this method appropriate for detection of these pathogens in naturally contaminated food samples.

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          High-resolution genotyping by amplicon melting analysis using LCGreen.

          Carl T. Wittwer, Gudrun H. Reed, Cameron N. Gundry … (2003)
          High-resolution amplicon melting analysis was recently introduced as a closed-tube method for genotyping and mutation scanning (Gundry et al. Clin Chem 2003;49:396-406). The technique required a fluorescently labeled primer and was limited to the detection of mutations residing in the melting domain of the labeled primer. Our aim was to develop a closed-tube system for genotyping and mutation scanning that did not require labeled oligonucleotides. We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), beta-globin (hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C, I507del) gene. PCR was performed in the presence of the double-stranded DNA dye LCGreen, and high-resolution amplicon melting curves were obtained. After fluorescence normalization, temperature adjustment, and/or difference analysis, sequence alterations were distinguished by curve shape and/or position. Heterozygous DNA was identified by the low-temperature melting of heteroduplexes not observed with other dyes commonly used in real-time PCR. The six common beta-globin genotypes (AA, AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon. The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp fragment that split into two melting domains. Because melting curve acquisition required only 1-2 min, amplification and analysis were achieved in 10-20 min with rapid cycling conditions. High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants. The technique requires only the usual unlabeled primers and a generic double-stranded DNA dye added before PCR for amplicon genotyping, and is a promising method for mutation screening.
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            Review of Salmonella detection and identification methods: Aspects of rapid emergency response and food safety

            John Hsieh, Kyung-Min Lee, Mick Runyon … (2015)
            Article 0 comments Cited 77 times – based on 0 reviews     Review now
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              Mechanism of Shiga Toxin Clustering on Membranes

              Weria Pezeshkian, Haifei Gao, Senthil Arumugam … (2016)
              The bacterial Shiga toxin interacts with its cellular receptor, the glycosphingolipid globotriaosylceramide (Gb3 or CD77), as a first step to entering target cells. Previous studies have shown that toxin molecules cluster on the plasma membrane, despite the apparent lack of direct interactions between them. The precise mechanism by which this clustering occurs remains poorly defined. Here, we used vesicle and cell systems and computer simulations to show that line tension due to curvature, height, or compositional mismatch, and lipid or solvent depletion cannot drive the clustering of Shiga toxin molecules. By contrast, in coarse-grained computer simulations, a correlation was found between clustering and toxin nanoparticle-driven suppression of membrane fluctuations, and experimentally we observed that clustering required the toxin molecules to be tightly bound to the membrane surface. The most likely interpretation of these findings is that a membrane fluctuation-induced force generates an effective attraction between toxin molecules. Such force would be of similar strength to the electrostatic force at separations around 1 nm, remain strong at distances up to the size of toxin molecules (several nanometers), and persist even beyond. This force is predicted to operate between manufactured nanoparticles providing they are sufficiently rigid and tightly bound to the plasma membrane, thereby suggesting a route for the targeting of nanoparticles to cells for biomedical applications.
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                Author and article information

                Contributors
                Afshin Akhondzadeh Basti:
                ORCID: https://orcid.org/0000-0002-8532-5757
                aakhond@ut.ac.ir
                Journal
                Journal ID (nlm-ta): Food Sci Nutr
                Journal ID (iso-abbrev): Food Sci Nutr
                Journal ID (doi): 10.1002/(ISSN)2048-7177
                Journal ID (publisher-id): FSN3
                Title: Food Science & Nutrition
                Publisher: John Wiley and Sons Inc. (Hoboken )
                ISSN (Electronic): 2048-7177
                Publication date (Electronic): 28 May 2020
                Publication date Collection: July 2020
                Volume: 8
                Issue: 7 ( doiID: 10.1002/fsn3.v8.7 )
                Pages: 3665-3672
                Affiliations
                [ 1 ] Department of Food Hygiene and Quality of Control Faculty of Veterinary Medicine University of Tehran Tehran Iran
                [ 2 ] Pediatric Infections Research Center Research Institute of children’s Health Shahid Beheshti University of Medical Sciences Tehran Iran
                Author notes
                [*] [* ] Correspondence

                Afshin Akhondzadeh Basti, Department of Food Hygiene, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. P.O. Box: 14155‐6453.

                Email: aakhond@ 123456ut.ac.ir

                Author information
                https://orcid.org/0000-0003-1412-0890
                https://orcid.org/0000-0002-8532-5757
                Article
                Publisher ID: FSN31649
                DOI: 10.1002/fsn3.1649
                PMC ID: 7382201
                SO-VID: 23f44601-c841-483e-97ae-fa61fdb2ed4d
                Copyright © © 2020 The Authors. Food Science & Nutrition published by Wiley Periodicals LLC.
                License:

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                Date received : 14 March 2020
                Date revision received : 26 April 2020
                Date accepted : 27 April 2020
                Page count
                Figures: 6, Tables: 1, Pages: 8, Words: 5077
                Funding
                Funded by: National Institute for Medical Research Development
                Award ID: 964212
                Categories
                Subject: Original Research
                Subject: Original Research
                Custom metadata
                source-schema-version-number 2.0
                cover-date July 2020
                details-of-publishers-convertor Converter:WILEY_ML3GV2_TO_JATSPMC version:5.8.5 mode:remove_FC converted:25.07.2020

                Keywords: escherichia coli o157: h7,food sample,hrma,shigella dysenteriae type 1,stx1a gene
                Data availability:
                Keywords: escherichia coli o157: h7, food sample, hrma, shigella dysenteriae type 1, stx1a gene

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