US EPA

7099864 

Journal Article 

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 

Shams, S; Bakhshi, B; Moghadam, TT; , 

2016 

1 

Jundishapur Journal of Microbiology
ISSN: 2008-3645
EISSN: 2008-4161 

AHVAZ JUNDISHAPUR UNIV MED SCI 

AHVAZ 

27127589 

10.5812/jjm.29645 

WOS:000373927000006 

Background: Campylobacteriosis is a zoonotic infectious disease caused by Corn pylobcicterjejuni and C coil.. The caclFgene 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 cuff gene and to develop a duplex PCRassay for simultaneous detection of C. colt and C.jejani, 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 Canipylobacter 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 Cjejuni and C. coil. The duplex PCRdesigned in this study showed that it could simultaneously detect and differentiate both C. jejuni and C. coif 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 mu g/mL for C.jejuni and C coli, respectively.Conclusions: In silico analysis of the cad!" full-gene showed variations between the two species that can be used as a molecular target for differentiating C jejimi and C coil in a single-step duplex-PCRassay with high specificity and sensitivity.