Genotype Variation in H. Pylori Isolates from Iranian Patients by RAPD-PCR

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1 Genotype Variation in H. Pylori Isolates by RAPD-PCR Genotype Variation in H. Pylori Isolates from Iranian Patients by RAPD-PCR Siavoshi F Department of Microbiology, Faculty of Science, Tehran University Shokouhfard M Department of Microbiology, Faculty of Science, Tehran University Malekzadeh R Digestive Disease Research Center, Tehran University of Medical Sciences Dinparast Jadid N Department of Biotechnology, Pasteur Institute of Iran, Tehran Massarrat S Digestive Disease Research Center, Tehran University of Medical Sciences Emrani A Department of Microbiology, Faculty of Science, Tehran University Corresponding Author: Farideh Siavoshi PhD, Department of Microbiology, Faculty of Science, Tehran University, Tehran, Iran. Tel: Fax: siavoshi@khayam.ut.ac.ir ABSTRACT Introduction and Aims: Do H. pylori isolates from normal and dyspeptic patients have similar genetic profiles? Since genotype variation occurs within H. pylori population with high frequency, it is tempting to exploit techniques such as RAPD-PCR to examine the possible correlation between specific H. pylori genotypes and different peptic diseases. In this study, H. pylori isolates from different dyspeptic patients were genotyped by RAPD-PCR. Materials and Methods: H. pylori isolates from 66 patients, 41 normal, 21 with ulcer, and 4 with cancer were cultured, DNAs were extracted by phenol-chloroform. RAPD-PCR was optimized, using 10-nt primers of arbitrary sequences (1281, 1254, 1247) and isolate-specific fingerprints were generated. Analysis of PCR products on agarose gel was performed using NTSYSpc program. Dendrograms were calculated according to Jaccard & Nei. Results: According to differences in genetic profiles, H. pylori isolates were clustered into 4 distinct groups: 2 groups consisted of isolates from normal patients, 2 groups of isolates from patients with ulcer, and isolates from patients with cancer were clustered along with isolates from normal and ulcer patients. Furthermore, isolates from ulcer patients appeared in the cluster related to isolates from normal patients. Conclusions: Genetic variation is quite frequent within H. pylori populations; thus RAPD-PCR is an effective technique to reveal genetic diversity of isolates from different dyspeptic patients. In this study, H. pylori strains were clustered into 4 groups: 2 groups from normal patients, and 2 from patients with ulcer. Further studies in larger populations will help to correlate a certain peptic disease to specific strain of H. pylori. Govaresh 2004; 9: 11-7 Keywords: H. pylori, Genetic diversity, RAPD-PCR, Dyspepsia diseases Govaresh\ Vol. 9 \ No. 1 \ Spring

2 - : RAPD-PCR H. pylori H. pylori RAPD-PCR H. pylori. RAPD-PCR H. pylori. DNA. ( ) H. pylori. ( ) RAPD -. (Fingerprinting) PCR. Jaccard & Nei NTSYSpc : H. pylori ( )... RAPD-PCR H. Pylori. H. pylori.. H. pylori. - : RAPD-PCR H. pylori : H. pylori ().. () CagA VacA () (H. pylori) (). - : : : siavoshi@khayam.ut.ac.ir / 1 / /

3 . ) (.. - ( CO2 %)... DNA DNA. - DNA ph= 3 TE (Sigma) SDS (Sigma). RNAse A (Roche) (20 mg/ml) K. /. DNA (ph=) TE DNA. % DNA.. RAPD-PCR. (Primme, Italy) 3. Tris-EDTA (). () () () H. pylori (). 1. H. pylori H. pylori. (-). H. pylori. ( ) (-). H. pylori Ribotyping RFLP.. RAPD-PCR PFGE REP-PCR H. pylori RAPD-PCR RAPD-PCR. H. pylori. H. pylori () (-) ). ) (. ( PCR 2.. H. pylori 1. heterogeneity 2. genetic fingerprint / / /

4 RAPD-PCR H. pylori 3000 bp : :- : Kb : bp : bp : RAPD-PCR :- : Kb : bp : :- ( ) H. pylori. annealing 5'-AACGCGCAAC-3' PCR. ( C) MgCl 2 (fermentas) dntp (Roche) Taq polymerase (fermentas) (). u MgCl 2 DNA. dntp Taq C) : PCR C) ( C C C ( C C annealing PCR.. % / PCR. NTSYSpc, version Jaccard & Nei ~2000 bp PCR DNA. PCR. ( ) -. ( ) -.( ) : RAPD-PCR Kb : :- bp : / / /

5 : NTSYS pc.. (I,II,III,IV) :C :U :N / / /

6 RAPD-PCR H. pylori... H. pylori DNA H. pylori... RAPD-PCR - H. pylori. - H. pylori. %/± /. RAPD-PCR. H. pylori H. pylori.. H. pylori H. pylori.. H. pylori.. H. pylori.. H. pylori. RAPD-PCR. H. pylori. Smith. RFLP RAPD H. pylori UreCD UreA 4. RAPD H. pylori RAPD-PCR. Akopyanz.. H. pylori. RAPD Akopyanz 4. typeability / / /

7 . :Vac A (Vacuolating cytotoxin A) H. pylori V-ATPase - Vac A. Vac A. C (signal, s) Vac A. ) s1 (middle, m). m2 m1 s2 (s1a, s1b, s1c s1/m1 s1/m2 H. pylori. s2/m2 CagA Vac As1. :CagA (Cytotoxin associated gene A) CagA. (Pathogenicity Island, PAI) (IV ) CagA CagA... CagA H. pylori CagA / /.. CagA :( ) Dendrogram. : -N (-) β.. DNA :Annealing.. H. pylori. :Genetic fingerprinting. DNA :Recombination DNA. :Heterogeneity. :RAPD-PCR (Random amplified polymorphism DNA) PCR DNA ( ).. :RFLP (Restriction fragment length polymorphism) ().. DNA :REP-PCR (Repetitive extragenic palindromic) PCR. :PFGE (Pulsed- field gel electrophoresis) DNA DNA.. DNA :Phenotype ). ( RNA :Ribotyping / / /

8 RAPD-PCR H. pylori 1. Massarrat S, Saberi-Firoozi M, Soleimani A et al. Peptic ulcer disease, irritable bowel syndrome and constipation in two populations in iran. Eur J Gastroenterol Hepatol. 1995; 7: Frenck RW Jr, Clemens J. Helicobacter in the developing world.microbes Infect. 2003; 5: Blaser MJ. The versatility of Helicobacter pylori in the adaptation to the human stomach. J Physiol Pharmacol ;48: Azuma T, Yamakawa A, Yamazaki S et al. Distinct diversity of the cag pathogenicity island among Helicobacter pylori strains in Japan. J Clin Microbiol. 2004; 42: Nomura A, Stemmermann GN, Chyou PH et al. Helicobacter pylori infection and the risk for duodenal and gastric ulceration. Ann Intern Med. 1994; 120: Del Giudice G, Michetti P. Inflammation, immunity and vaccines for Helicobacter pylori. Helicobacter. 2004; 9 (Suppl 1): Lee KH, Cho MJ, Yamaoka Y et al. Alanine-threonine polymorphism of Helicobacter pylori RpoB is correlated with differential induction of interleukin-8 in MKN45 cells. J Clin Microbiol. 2004; 42: Go MF, Graham DY. How does Helicobacter pylori cause duodenal ulcer disease: the bug, the host, or both? J Gastroenterol Hepatol. 1994; 9 (Suppl 1): S Hazell Sl, Andrews Rh, Mitchell HM et al. Genetic relationship among isolates of Helicobacter pylori: evidence for the existence of a Helicobacter pylori species-complex. FEMS Microbiol Lett. 1997; 150: Marshall DG, Dundon WG, Beesley SM et al. Helicobacter pylori--a conundrum of genetic diversity. Microbiology. 1998; 144: Logan RP, Berg DE. Genetic diversity of Helicobacter pylori. Lancet. 1996; 348: Taylor NS, Fox JG, Akopyants NS et al. Long-term colonization with single and multiple strains of Helicobacter pylori assessed by DNA fingerprinting. J Clin Microbiol. 1995; 33: Akopyanz N, Bukanov NO, Westblom TU et al. PCRbased RFLP analysis of DNA sequence diversity in the gastric pathogen Helicobacter pylori. Nucleic Acids Res. 1992; 20: Desai M, Linton D, Owen RJ et al. Genetic diversity of Helicobacter pylori indexed with respect to clinical symptomatology, using a 16S rrna and a speciesspecific DNA probe. J Appl Bacteriol. 1993; 75: Go MF, Chan KY, Versalovic J et al. Cluster analysis of Helicobacter pylori genomic DNA fingerprints suggests gastroduodenal disease-specific associations. Scand J Gastroenterol. 1995; 30: Salama SM, Jiang Q, Chang N et al. Characterization of chromosomal DNA profiles from Helicobacter pylori strains isolated from sequential gastric biopsy specimens. J Clin Microbiol. 1995; 33: Berg DE, Akopyants NS, Kersulyte D. Fingerprinting microbial genome using the RAPD or AP-PCR method. Methods Molecular Cell Biol.1994; 5: Yakoob J, Hu GL, Fan XG, et al. Diversity of Helicobacter pylori among Chinese persons with H. pylori infection. APMIS. 2000; 108: Kuo CH, Poon SK, Su YC et al. Heterogeneous Helicobacter pylori isolates from H. pylori-infected couples in Taiwan. J Infect Dis. 1999; 180: Karita M, Teramukai S, Matsumoto S. Risk of Helicobacter pylori transmission from drinking well water is higher than that from infected intrafamilial members in Japan. Dig Dis Sci. 2003; 48: Kim JW, Kim JG, Chae SL et al. High prevalence of multiple strain colonization of Helicobacter pylori in Korean patients: DNA diversity among clinical isolates from the gastric corpus, antrum and duodenum. Korean J Intern Med. 2004; 19: Power EG.RAPD typing in microbiology - a technical review. J Hosp Infect. 1996; 34: Bush U, Nitschko H. Methods for the differentiation of microorganisms. J Chromatogr B Biomed Sci Appl. 1999; 722: Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 1990; 24: Akopyanz N, Bukanov NO, Westblom TU et al. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res. 1992; 20: Smith S, Cantet F, Angelini F et al. Discriminatory power of RAPD, PCR-RFLP and southern blot analyses of urecd or urea gene probes on Helicobacter pylori isolates. Z Naturforsch [C]. 2002; 57: Kidd M, Atherton JC, Lastovica AJ et al. Clustering of South African Helicobacter pylori isolates from peptic ulcer disease patients is demonstrated by repetitive extragenic palindromic-pcr Kid fingerprinting. J Clin Microbiol. 2001; 39: Swaminathan B, Barrett TJ. Amplification methods for epidemiologic investigations of infectious disease. J Micro Methods. 1995; 23: / / /

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