title: 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.
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.
  
  
  OBJECTIVE
  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.
  
  
  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.
authors.affiliation: Department of Medical Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran.
authors.forename: Saeed
authors.initials: S
authors.lastname: Shams
authors.affiliation: Department of Medical Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, IR Iran.
authors.forename: Bita
authors.initials: B
authors.lastname: Bakhshi
authors.affiliation: Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IR Iran.
authors.forename: Tahereh
authors.initials: T
authors.lastname: Tohidi Moghadam
keywords: C. coli; Campylobacter jejuni; Duplex PCR; In Silico; cadF