Designing a Duplex Polymerase Chain Reaction Assay for Specific Detection of Campylobacter jejuni and C. coli using analysis of enzymatic digestion pattern.ģ.3. This fragment and other selected sequences from the full gene of cadF were subjected to in silico analysis with the online NEB cutter program () to compare and select a proper restriction endonuclease for discriminating between C. ( 15) as a specific gene for detection of Campylobacter spp., was used as a reference sequence in this study. The conserved internal fragment (400 bp) of the cadF gene, reported by Konkel et al. jejuni 81116, complete genomeĬampylobacter coli RM2228 cont193, whole genome shotgun sequenceĬampylobacter coli RM1875, complete genomeĬampylobacter coli 15-537360, complete genomeĬampylobacter coli RM5611, complete genomeĬampylobacter coli CVM N29710, complete genomeĬampylobacter coli RM4661, complete genomeĬampylobacter coli JV20 contig00034, whole genome shotgun sequenceĬampylobacter coli JV20 genomic scaffold SCAFFOLD1, whole genome shotgun sequence jejuni NCTC 11168 complete genomeĬampylobacter jejuni RM1221, complete genomeĬampylobacter jejuni subsp. jejuni PT14, complete genomeĬampylobacter jejuni subsp. jejuni 00-2544, complete genomeĬampylobacter jejuni subsp. jejuni 00-2538, complete genomeĬampylobacter jejuni subsp. jejuni strain MTVDSCj20, complete genomeĬampylobacter jejuni subsp. Multiple alignments were performed using the CLC sequence viewer 7.6 software (CLC bio, Aarhus, Denmark).Ĭampylobacter jejuni subsp. coli were acquired from NCBI GenBank () ( Table 1). The cadF sequences from the complete genome of C. jejuni, the two commonly encountered species in human Campylobacteriosis.ģ. The aim of this study was to analyze the cadF gene and to develop and evaluate a single-step duplex polymerase chain reaction (PCR) assay for simultaneous detection of C. There is no documented bioinformatics study on the cadF gene full-sequence analysis in C. In most studies a fragment from this gene with a length of 400 bp is used for identification of Campylobacter spp. Among them, the cadF gene encodes a fibronectin-binding protein that promotes bacteria-host cell interaction and has been described as a conserve and genus-specific gene. asp, hipO, ceuE, cadF, 16SrRNA, 23S rRNA and cdt, fur, glyA, cdtABC, ceuB–E and fliY) (9). In molecular methods different genetic targets have been used for the detection Campylobacter species (e.g. Moreover, the emergence of viable but non-culturable (VBNC) phenotypes should not be ignored.ĭifferentiation of the two species is only performed through hippurate hydrolysis biochemical test or molecular-based detections ( 7- 10). jejuni however, the culture conditions for detection of these fastidious bacteria are complicated and time consuming, which in some cases make the recovery of bacteria unsuccessful. Culture is the gold standard of diagnosis of C. Diagnosis of campylobacteriosis is performed through microbiological, molecular and serological tests. These complications can be prevented or lowered with rapid and accurate detection of etiological agents of the disease. Although, in most cases the illness is self-limited and rarely fatal yet post-infectious acute immune-mediated neurologic complications such as Guillain-Barre syndrome and Miller Fisher syndrome can occur, which are the consequence of molecular mimicry between lipooligosaccharides (LOS) of bacterial cell wall and gangliosides in peripheral nerves of humans ( 5, 6). The symptoms of campylobacteriosis can vary from mild to severe complications, including abdominal pain, fever, myalgia and watery or bloody diarrhea. Although, poultry and poultry products are important sources of Campylobacteriosis, yet the organism can be transmitted to humans via contact with other warm-blooded animals such as cattle, pigs, sheep, ostriches, shellfish, and pets ( 3, 4). coli have been recognized as the most common causes of bacterial diarrhea in humans, especially among children less than five years of age and young adults ( 2). BackgroundĬampylobacter enteritis is one of the most frequent food-borne infections worldwide ( 1). In Silico Duplex PCR cadF Campylobacter jejuni C. coli in a single-step duplex-PCR assay with high specificity and sensitivity. 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.
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