Abstract
Campylobacter spp. are the main cause of human gastroenteritis. The 16SrRNA sequencing is one of fast molecualr method to detect this fastidious. In this study, we compared the sequencing of 16srRNA genewith four housekeeping genestodetect Campylobacter spp. in patients with diarrhea and healthy people. 60 samples of Campylobacter DNA extracted from stool samples of 30 patients with diarrhea and 30 healthy people were used. In order to detect Campylobacter, we designed primers for proliferation of 16SrRNA, cadF, dnaJ, slyD , and rpoA genes using Primer 3, Mega 4.0 and Blast software. Then the PCR products were sequenced using ABI system. The sequencing showed concordance of PCR-products with deposited sequences in the Gene Bank. Among diarrhea patients, 53.3% of samples were significantly (p< 0.05) positive for slyD and cadF genes and 50% of samples were positive using 16SrRNA, rpoA, and dnaJ genes by PCR assay. The average of sensitivity and specificity were found 53.33% and 83.33%, respectively. Due to various copies of repeated sequences of 16SrRNA gene, analyzing its amplicons on electrophoresis may be more difficult than the slyD and cadF genes. According to our results, among the 5 studied genes; the highest detection rate was related to slyD and cadF genes. Although, dnaJ and rpoA genes,instead of 16SrRNA gene, can be considered as appropriate genes for molecular detection of Campylobacter bacteria.
Highlights
Campylobacter spp. are gram-negative, spiral, and coma-shape bacteria
The purpose of this study is to compare the detection of Campylobacter using the 16SrRNA gene with slyD, cadF, rpoA, and dnaJ genes for determination in patients with diarrhea admitted to Amirkabir Hospital in Arak and healthy individuals people referred to the Arak health center in 2017-2018
Polymerase chain reaction (PCR) results of housekeeping genes Among of 60 samples, 21 samples (35%), 21 samples (35%), 20 samples (33.33%), samples (33.33%), 20 samples (33.33%), and 20 samples (33.33%) were positive for Campylobacter by slyD, cadF, dnaJ, rpoA, and 16SrRNA, respectively Table 2)
Summary
Campylobacter spp. are gram-negative, spiral, and coma-shape bacteria. Campylobacter spp. require microaerophilic conditions (10% CO2, 5% O2) and temperatures 37- 42 °C to grow [1]. The copy numbers can differs between Campylobacter spp. and only a limited number of bacterial genomes have one copy of 16SrRNA gene that its varies may be simultaneous with increasing number of copies [11]. The sequence of 16SrRNA gene can be different among bacteria communities [12]. We compared the sequencing of 16srRNA genewith four housekeeping genestodetect Campylobacter spp. in patients with diarrhea and healthy people. In order to detect Campylobacter, we designed primers for proliferation of 16SrRNA, cadF, dnaJ, slyD, and rpoA genes using Primer 3, Mega 4.0 and Blast software. 53.3% of samples were significantly (p< 0.05) positive for slyD and cadF genes and 50% of samples were positive using 16SrRNA, rpoA, and dnaJ genes by PCR assay. DnaJ and rpoA genes, instead of 16SrRNA gene, can be considered as appropriate genes for molecular detection of Campylobacter bacteria
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