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      In Silico Analysis of the cadF Gene and Development of a Duplex Polymerase Chain Reaction for Species-Specific Identification of Campylobacter jejuni and Campylobacter coli

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          Abstract

          Background

          Campylobacteriosis is a zoonotic infectious disease caused by Campylobacter jejuni and C. coli. The cadF gene is considered as a genus-specific gene while other genes are mainly used for discrimination at the species level.

          Objectives

          This study aimed to analyze the cadF gene and to develop a duplex PCR assay for simultaneous detection of C. coli and C. jejuni, the two commonly encountered species.

          Materials and Methods

          In silico analysis of the cadF gene was carried out by several software and available online tools. A duplex PCR optimized with specific primers was used for detection and differentiation of both species. To evaluate specificity and sensitivity of the test, a panel of different Campylobacter spp. together with several intestinal bacterial pathogens was tested. The limit of detection (LOD) of method was determined using serial dilutions of standard genomes.

          Results

          The analysis of the full size cadF gene indicated variations in this gene, which can be used to differentiate C. jejuni and C. coli. The duplex PCR designed in this study showed that it could simultaneously detect and differentiate both C. jejuni and C. coli with product sizes of 737 bp and 461 bp, respectively. This assay, with 100% specificity and sensitivity, had a limit of detection (LOD) of about 14 and 0.7 µg/mL for C. jejuni and C. coli, respectively.

          Conclusions

          In silico analysis of the cadF full-gene showed variations between the two species that can be used as a molecular target for differentiating C. jejuni and C. coli in a single-step duplex-PCR assay with high specificity and sensitivity.

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          Most cited references24

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          Colony multiplex PCR assay for identification and differentiation of Campylobacter jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus subsp. fetus.

          G Wang, C G Clark, T. M. Taylor … (2002)
          A multiplex PCR assay was used to simultaneously detect genes from the five major clinically relevant Campylobacter spp. Those genes selected were hipO and 23S rRNA from Campylobacter jejuni; glyA from each of C. coli, C. lari, and C. upsaliensis; and sapB2 from C. fetus subsp. fetus. The assay was evaluated with 137 clinical and environmental isolates and was found to be rapid and easy to perform and had a high sensitivity and specificity for characterizing isolates, even in mixed cultures.
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            Differentiation of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis by a multiplex PCR developed from the nucleotide sequence of the lipid A gene lpxA.

            John Klena, Craig Parker, J Ibbitt … (2004)
            We describe a multiplex PCR assay to identify and discriminate between isolates of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis. The C. jejuni isolate F38011 lpxA gene, encoding a UDP-N-acetylglucosamine acyltransferase, was identified by sequence analysis of an expression plasmid that restored wild-type lipopolysaccharide levels in Escherichia coli strain SM105 [lpxA(Ts)]. With oligonucleotide primers developed to the C. jejuni lpxA gene, nearly full-length lpxA amplicons were amplified from an additional 11 isolates of C. jejuni, 20 isolates of C. coli, 16 isolates of C. lari, and five isolates of C. upsaliensis. The nucleotide sequence of each amplicon was determined, and sequence alignment revealed a high level of species discrimination. Oligonucleotide primers were constructed to exploit species differences, and a multiplex PCR assay was developed to positively identify isolates of C. coli, C. jejuni, C. lari, and C. upsaliensis. We characterized an additional set of 41 thermotolerant isolates by partial nucleotide sequence analysis to further demonstrate the uniqueness of each species-specific region. The multiplex PCR assay was validated with 105 genetically defined isolates of C. coli, C. jejuni, C. lari, and C. upsaliensis, 34 strains representing 12 additional Campylobacter species, and 24 strains representing 19 non-Campylobacter species. Application of the multiplex PCR method to whole-cell lysates obtained from 108 clinical and environmental thermotolerant Campylobacter isolates resulted in 100% correlation with biochemical typing methods.
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              Identification of the enteropathogens Campylobacter jejuni and Campylobacter coli based on the cadF virulence gene and its product.

              Elin Gray, J.-G. Yoon, S Garvis … (1999)
              Campylobacter jejuni and Campylobacter coli are common causes of gastroenteritis in humans. Infection with C. jejuni or C. coli is commonly acquired by eating undercooked chicken. The goal of this study was to develop specific detection assays for C. jejuni and C. coli isolates based on the cadF virulence gene and its product. The cadF gene from C. jejuni and C. coli encodes a 37-kDa outer membrane protein that promotes the binding of these pathogens to intestinal epithelial cells. A fragment of approximately 400 bp was amplified from 38 of 40 (95%) C. jejuni isolates and 5 of 6 (83.3%) C. coli isolates with primers designed to amplify an internal fragment of the cadF gene. PCR was then used to amplify Campylobacter DNA from store-bought chickens. A 400-bp band was amplified from 26 of the 27 chicken carcasses tested by the PCR-based assay. The CadF protein was detected in every C. jejuni and C. coli isolate tested, as judged by immunoblot analysis with a rabbit anti-C. jejuni 37-kDa serum. In addition, methanol-fixed samples of whole-cell C. jejuni and C. coli were detected with the rabbit anti-37-kDa serum by using an indirect-immunofluorescence microscopy assay. These findings indicate that the cadF gene and its product are conserved among C. jejuni and C. coli isolates and that a PCR assay based on the cadF gene may be useful for the detection of Campylobacter organisms in food products.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Jundishapur J Microbiol
                Journal ID (iso-abbrev): Jundishapur J Microbiol
                Journal ID (doi): 10.5812/jjm
                Journal ID (publisher-id): Kowsar
                Title: Jundishapur Journal of Microbiology
                Publisher: Kowsar
                ISSN (Print): 2008-3645
                ISSN (Electronic): 2008-4161
                Publication date Collection: February 2016
                Publication date (Electronic): 09 February 2016
                Volume: 9
                Issue: 2
                Electronic Location Identifier: e29645
                Affiliations
                [1 ]Department of Medical Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran
                [2 ]Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IR Iran
                Author notes
                [* ]Corresponding author: Bita Bakhshi, Department of Medical Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran. Tel: +98-2182884558, Fax: +98-2182884555, E-mail: b.bakhshi@ 123456modares.ac.ir
                Article
                DOI: 10.5812/jjm.29645
                PMC ID: 4842252
                PubMed ID: 27127589
                SO-VID: 2d95b68f-8e75-4371-938d-fabc1e1b5222
                Copyright © Copyright © 2016, Ahvaz Jundishapur University of Medical Sciences
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License ( http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited.

                History
                Date received : 04 May 2015
                Date revision received : 28 September 2015
                Date accepted : 03 November 2015
                Categories
                Subject: Research Article

                Keywords: in silico,duplex pcr,cadf,campylobacter jejuni,c. coli
                Data availability:
                Keywords: in silico, duplex pcr, cadf, campylobacter jejuni, c. coli

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