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.

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.

Keywords: C. coli; Campylobacter jejuni; Duplex PCR; In Silico; cadF.

Figure 1.. Position of Primers for Developing the Duplex Polymerase Chain Reaction Assay

Black thin lines of 999 and 960 bp are related to the cadF gene of C. coli and C. jejuni, respectively. The discontinuity is related to deletion in C. jejuni. A, produced a 400-bp fragment from both C. jejuni and C. coli (as the positive control of the assay); B, produced a 461-bp fragment from C. coli; C, produced a 737-bp fragment from C. jejuni.

Figure 2.. Analysis and Multiple Alignment of the cadF Complete Gene of Campylobacter jejuni (960 bp) and C. coli (999 bp) by the CLC Software

Dashes in the lower box indicate deleted nucleotides in C. jejuni. The blue box is the conserved 400-bp internal region introduced by Konkel et al.

Figure 3.. Agarose Gel Electrophoresis of the Duplex Polymerase Chain Reaction Assay with Specific Primers

Lanes 1 and 2, 400-bp fragment of the cadF gene of Campylobacter jejuni and Campylobacter coli, respectively as positive controls; lanes 4 - 6, 737 bp fragment of C. jejuni; lanes 9 - 11, 461 bp fragment of C. coli; lanes 3, 7, 8, 12 and 13, negative controls; lane M, 1 kb molecular weight marker.