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Ghorbani marghmaleki E, Ahmadi A, Arjomandzadegan M, Akbari M, Karamghoshchi A. Molecular Detection of Campylobacter Species: Comparision of 16SrRNA with slyD, cadF, rpoA, and dnaJ Sequencing. rbmb.net 2020; 9 (3) :257-263
URL: http://rbmb.net/article-1-501-en.html
URL: http://rbmb.net/article-1-501-en.html
Elahe Ghorbani marghmaleki 
, Azam Ahmadi * , Mohammad Arjomandzadegan , Majid Akbari , Aysan Karamghoshchi
, Azam Ahmadi * , Mohammad Arjomandzadegan , Majid Akbari , Aysan Karamghoshchi
Infectious Diseases Research Center (IDRC), Arak University of Medical Sciences, Arak, Iran.
Abstract: (3250 Views)
Background: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
Methods: 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.
Results: 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.
Conclusions: 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.
Type of Article: Original Article |
Subject:
Microbiology
Received: 2020/05/19 | Accepted: 2020/06/8 | Published: 2020/12/1
Received: 2020/05/19 | Accepted: 2020/06/8 | Published: 2020/12/1
References
1. Silva J, Leite D, Fernandes M, Mena C, Gibbs PA, et al. Campylobacter spp. as a foodborne pathogen: a review. Front Microbiol. 2011;2:200. [DOI:10.3389/fmicb.2011.00200] [PMID] [PMCID]
2. Nuijten PJ, Bartels C, Bleumink-Pluym NM, Gaastra W, van der Zeijst BA. Size and physical map of the Campylobacter jejuni chromosome. Nucleic Acids Res. 1990;18(21):6211-6214. [DOI:10.1093/nar/18.21.6211] [PMID] [PMCID]
3. Altekruse SF, Stern NJ, Fields PI, Swerdlow DL. Campylobacter jejuni-an emerging foodborne pathogen. Emerg Infect Dis. 1999;5(1):28-35. [DOI:10.3201/eid0501.990104] [PMID] [PMCID]
4. Wassenaar TM. Toxin production by Campylobacter spp. Clin Microbiol Rev. 1997;10(3):466-476. [DOI:10.1128/CMR.10.3.466] [PMID] [PMCID]
5. Laprade N, Cloutier M, Lapen DR, Topp E, Wilkes G, Villemur R, et al. Detection of virulence, antibiotic resistance and toxin (VAT) genes in Campylobacter species using newly developed multiplex PCR assays. J Microbiol Methods. 2016;124:41-7. [DOI:10.1016/j.mimet.2016.03.009] [PMID]
6. Ritz M, Garenaux A, Berge M, Federighi M. Determination of rpoA as the most suitable internal control to study stress response in C. jejuni by RT-qPCR and application to oxidative stress. J Microbiol Methods. 2009;76(2):196-200. [DOI:10.1016/j.mimet.2008.10.014] [PMID]
7. Hugenholtz P, Goebel BM, Pace NR. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J bacteriol. 1998;180(18):4765-4774. [DOI:10.1128/JB.180.18.4765-4774.1998] [PMID] [PMCID]
8. Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U S A. 1985;82(20):6955-6959. [DOI:10.1073/pnas.82.20.6955] [PMID] [PMCID]
9. Ward DM, Weller R, Bateson MM. 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature. 1990;345(6270):63-5. [DOI:10.1038/345063a0] [PMID]
10. Klappenbach JA, Saxman PR, Cole JR, Schmidt TM. rrndb: the ribosomal RNA operon copy number database. Nucleic Acids Res. 2001;29(1):181-4. [DOI:10.1093/nar/29.1.181] [PMID] [PMCID]
11. Acinas SG, Marcelino LA, Klepac-Ceraj V, Polz MF. Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons. J Bacteriol. 2004;186(9):2629-35. [DOI:10.1128/JB.186.9.2629-2635.2004] [PMID] [PMCID]
12. Case RJ, Boucher Y, Dahllöf I, Holmström C, Doolittle WF, Kjelleberg S. Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies. Appl Environ Microbiol. 2007;73(1):278-88. [DOI:10.1128/AEM.01177-06] [PMID] [PMCID]
13. Leblanc-Maridor M, Garénaux A, Beaudeau F, Chidaine B, Seegers H, Denis M, et al. Quantification of Campylobacter spp. in pig feces by direct real-time PCR with an internal control of extraction and amplification. J Microbiol Methods. 2011;85(1):53-61. [DOI:10.1016/j.mimet.2011.01.013] [PMID]
14. Kang CR, Bang JH, Cho S-I. Campylobacter jejuni Foodborne Infection Associated with Cross-contamination: Outbreak in Seoul in 2017. Infect Chemother. 2019;51(1):21-27. [DOI:10.3947/ic.2019.51.1.21] [PMID] [PMCID]
15. Bolton DJ. Campylobacter virulence and survival factors. Food microbiol. 2015;48:99-108. [DOI:10.1016/j.fm.2014.11.017] [PMID]
16. Bang DD, Scheutz F, Oren Gradel K, Nielsen E, Pedersen K, Engberg J, et al. Detection of seven virulence and toxin genes of Campylobacter jejuni and Campylobacter coli isolates from different sources and cytolethal distending toxin production suggest potential diversity of pathogenic properties among isolates. Genome Letters. 2003;2:62-72.
17. Konkel ME, Kim BJ, Rivera-Amill V, Garvis SG. Identification of proteins required for the internalization of Campylobacter jejuni intocultured mammalian cells. Adv Exp Med Biol. 1999;473:215-24. [DOI:10.1007/978-1-4615-4143-1_22] [PMID]
18. Datta S, Niwa H, Itoh K. Prevalence of 11 pathogenic genes of Campylobacter jejuni by PCR in strains isolated from humans, poultry meat and broiler and bovine faeces. J Med Microbiol. 2003;52(pt 4):345-348. [DOI:10.1099/jmm.0.05056-0] [PMID]
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