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. 1997 Dec;35(12):3021–3025. doi: 10.1128/jcm.35.12.3021-3025.1997

Differentiation of Helicobacter pylori strains directly from gastric biopsy specimens by PCR-based restriction fragment length polymorphism analysis without culture.

C Li 1, T Ha 1, D S Chi 1, D A Ferguson Jr 1, C Jiang 1, J J Laffan 1, E Thomas 1
PMCID: PMC230115  PMID: 9399487

Abstract

Recent studies have shown the usefulness of PCR-based restriction fragment length polymorphism (RFLP) analysis for differentiating Helicobacter pylori strains isolated by culture. For this study, a PCR-based RFLP assay was developed for directly typing H. pylori strains from gastric biopsy specimens. Nineteen gastric biopsy specimens obtained from patients undergoing endoscopy for gastrointestinal complaints were cultured for isolation of H. pylori. Genomic DNA preparations from these gastric biopsy specimens and the corresponding H. pylori isolates were tested by our PCR-based RFLP assay. The 1,179-bp H. pylori DNA fragments amplified by the PCR assay were digested with the restriction enzymes HhaI, MboI, and AluI and analyzed by agarose gel electrophoresis. HhaI, MboI, and AluI digestion produced 11, 10, and 6 distinguishable digestion patterns, respectively, from the 19 H. pylori isolates tested and generated 13, 11, and 6 different patterns, respectively, from the 19 gastric biopsy specimens. The patterns from 13 of the 19 gastric biopsy specimens matched those of the H. pylori isolates from the corresponding patients. The patterns from the remaining six biopsy specimens appeared to represent infection by two strains of H. pylori; the pattern of one strain was identical to that of the isolate from the corresponding patient. By combining all the restriction enzyme digestion patterns obtained by using HhaI, MboI, and AluI, we observed 19 distinct RFLP patterns from the 19 specimens. The results suggest that the PCR-based RFLP analysis method may be useful as a primary technique to identify and distinguish H. pylori strains directly from gastric biopsy specimens without culture of the organisms.

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Selected References

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  1. Akopyanz N., Bukanov N. O., Westblom T. U., Berg D. E. PCR-based RFLP analysis of DNA sequence diversity in the gastric pathogen Helicobacter pylori. Nucleic Acids Res. 1992 Dec 11;20(23):6221–6225. doi: 10.1093/nar/20.23.6221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akopyanz N., Bukanov N. O., Westblom T. U., Kresovich S., Berg D. E. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting. Nucleic Acids Res. 1992 Oct 11;20(19):5137–5142. doi: 10.1093/nar/20.19.5137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bickley J., Owen R. J., Fraser A. G., Pounder R. E. Evaluation of the polymerase chain reaction for detecting the urease C gene of Helicobacter pylori in gastric biopsy samples and dental plaque. J Med Microbiol. 1993 Nov;39(5):338–344. doi: 10.1099/00222615-39-5-338. [DOI] [PubMed] [Google Scholar]
  4. Burnie J. P., Lee W., Dent J. C., McNulty C. A. Immunoblot fingerprinting of Campylobacter pylori. J Med Microbiol. 1988 Oct;27(2):153–159. doi: 10.1099/00222615-27-2-153. [DOI] [PubMed] [Google Scholar]
  5. Clayton C. L., Kleanthous H., Dent J. C., McNulty C. A., Tabaqchali S. Evaluation of fingerprinting methods for identification of Helicobacter pylori strains. Eur J Clin Microbiol Infect Dis. 1991 Dec;10(12):1040–1047. doi: 10.1007/BF01984926. [DOI] [PubMed] [Google Scholar]
  6. Clayton C. L., Kleanthous H., Morgan D. D., Puckey L., Tabaqchali S. Rapid fingerprinting of Helicobacter pylori by polymerase chain reaction and restriction fragment length polymorphism analysis. J Clin Microbiol. 1993 Jun;31(6):1420–1425. doi: 10.1128/jcm.31.6.1420-1425.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Desai M., Linton D., Owen R. J., Stanley J. Molecular typing of Helicobacter pylori isolates from asymptomatic, ulcer and gastritis patients by urease gene polymorphism. Epidemiol Infect. 1994 Feb;112(1):151–160. doi: 10.1017/s0950268800057514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ferguson D. A., Jr, Li C., Patel N. R., Mayberry W. R., Chi D. S., Thomas E. Isolation of Helicobacter pylori from saliva. J Clin Microbiol. 1993 Oct;31(10):2802–2804. doi: 10.1128/jcm.31.10.2802-2804.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Figura N., Guglielmetti P., Rossolini A., Barberi A., Cusi G., Musmanno R. A., Russi M., Quaranta S. Cytotoxin production by Campylobacter pylori strains isolated from patients with peptic ulcers and from patients with chronic gastritis only. J Clin Microbiol. 1989 Jan;27(1):225–226. doi: 10.1128/jcm.27.1.225-226.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Forman D., Newell D. G., Fullerton F., Yarnell J. W., Stacey A. R., Wald N., Sitas F. Association between infection with Helicobacter pylori and risk of gastric cancer: evidence from a prospective investigation. BMJ. 1991 Jun 1;302(6788):1302–1305. doi: 10.1136/bmj.302.6788.1302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Foxall P. A., Hu L. T., Mobley H. L. Use of polymerase chain reaction-amplified Helicobacter pylori urease structural genes for differentiation of isolates. J Clin Microbiol. 1992 Mar;30(3):739–741. doi: 10.1128/jcm.30.3.739-741.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fujimoto S., Marshall B., Blaser M. J. PCR-based restriction fragment length polymorphism typing of Helicobacter pylori. J Clin Microbiol. 1994 Feb;32(2):331–334. doi: 10.1128/jcm.32.2.331-334.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Genta R. M., Hamner H. W., Graham D. Y. Gastric lymphoid follicles in Helicobacter pylori infection: frequency, distribution, and response to triple therapy. Hum Pathol. 1993 Jun;24(6):577–583. doi: 10.1016/0046-8177(93)90235-9. [DOI] [PubMed] [Google Scholar]
  14. Graham D. Y. Campylobacter pylori and peptic ulcer disease. Gastroenterology. 1989 Feb;96(2 Pt 2 Suppl):615–625. doi: 10.1016/s0016-5085(89)80057-5. [DOI] [PubMed] [Google Scholar]
  15. Labigne A., Cussac V., Courcoux P. Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity. J Bacteriol. 1991 Mar;173(6):1920–1931. doi: 10.1128/jb.173.6.1920-1931.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lage A. P., Godfroid E., Fauconnier A., Burette A., Butzler J. P., Bollen A., Glupczynski Y. Diagnosis of Helicobacter pylori infection by PCR: comparison with other invasive techniques and detection of cagA gene in gastric biopsy specimens. J Clin Microbiol. 1995 Oct;33(10):2752–2756. doi: 10.1128/jcm.33.10.2752-2756.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Langenberg W., Rauws E. A., Widjojokusumo A., Tytgat G. N., Zanen H. C. Identification of Campylobacter pyloridis isolates by restriction endonuclease DNA analysis. J Clin Microbiol. 1986 Sep;24(3):414–417. doi: 10.1128/jcm.24.3.414-417.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Li C., Ferguson D. A., Jr, Ha T., Chi D. S., Thomas E. A highly specific and sensitive DNA probe derived from chromosomal DNA of Helicobacter pylori is useful for typing H. pylori isolates. J Clin Microbiol. 1993 Aug;31(8):2157–2162. doi: 10.1128/jcm.31.8.2157-2162.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Li C., Ha T., Ferguson D. A., Jr, Chi D. S., Zhao R., Patel N. R., Krishnaswamy G., Thomas E. A newly developed PCR assay of H. pylori in gastric biopsy, saliva, and feces. Evidence of high prevalence of H. pylori in saliva supports oral transmission. Dig Dis Sci. 1996 Nov;41(11):2142–2149. doi: 10.1007/BF02071393. [DOI] [PubMed] [Google Scholar]
  20. Li C., Musich P. R., Ha T., Ferguson D. A., Jr, Patel N. R., Chi D. S., Thomas E. High prevalence of Helicobacter pylori in saliva demonstrated by a novel PCR assay. J Clin Pathol. 1995 Jul;48(7):662–666. doi: 10.1136/jcp.48.7.662. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lopez C. R., Owen R. J., Desai M. Differentiation between isolates of Helicobacter pylori by PCR-RFLP analysis of urease A and B genes and comparison with ribosomal RNA gene patterns. FEMS Microbiol Lett. 1993 Jun 1;110(1):37–43. doi: 10.1111/j.1574-6968.1993.tb06292.x. [DOI] [PubMed] [Google Scholar]
  22. Majewski S. I., Goodwin C. S. Restriction endonuclease analysis of the genome of Campylobacter pylori with a rapid extraction method: evidence for considerable genomic variation. J Infect Dis. 1988 Mar;157(3):465–471. doi: 10.1093/infdis/157.3.465. [DOI] [PubMed] [Google Scholar]
  23. Megraud F., Bonnet F., Garnier M., Lamouliatte H. Characterization of "Campylobacter pyloridis" by culture, enzymatic profile, and protein content. J Clin Microbiol. 1985 Dec;22(6):1007–1010. doi: 10.1128/jcm.22.6.1007-1010.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moore R. A., Kureishi A., Wong S., Bryan L. E. Categorization of clinical isolates of Helicobacter pylori on the basis of restriction digest analyses of polymerase chain reaction-amplified ureC genes. J Clin Microbiol. 1993 May;31(5):1334–1335. doi: 10.1128/jcm.31.5.1334-1335.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Owen R. J., Bickley J., Costas M., Morgan D. R. Genomic variation in Helicobacter pylori: application to identification of strains. Scand J Gastroenterol Suppl. 1991;181:43–50. doi: 10.3109/00365529109093207. [DOI] [PubMed] [Google Scholar]
  26. Owen R. J., Bickley J., Hurtado A., Fraser A., Pounder R. E. Comparison of PCR-based restriction length polymorphism analysis of urease genes with rRNA gene profiling for monitoring Helicobacter pylori infections in patients on triple therapy. J Clin Microbiol. 1994 May;32(5):1203–1210. doi: 10.1128/jcm.32.5.1203-1210.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Penfold S. S., Lastovica A. J., Elisha B. G. Demonstration of plasmids in Campylobacter pylori. J Infect Dis. 1988 Apr;157(4):850–851. doi: 10.1093/infdis/157.4.850. [DOI] [PubMed] [Google Scholar]
  28. Prewett E. J., Bickley J., Owen R. J., Pounder R. E. DNA patterns of Helicobacter pylori isolated from gastric antrum, body, and duodenum. Gastroenterology. 1992 Mar;102(3):829–833. doi: 10.1016/0016-5085(92)90165-u. [DOI] [PubMed] [Google Scholar]
  29. Romero Lopez C., Owen R. J., Banatvala N., Abdi Y., Hardie J. M., Davies G. R., Feldman R. Comparison of urease gene primer sequences for PCR-based amplification assays in identifying the gastric pathogen Helicobacter pylori. Mol Cell Probes. 1993 Dec;7(6):439–446. doi: 10.1006/mcpr.1993.1065. [DOI] [PubMed] [Google Scholar]
  30. Simor A. E., Shames B., Drumm B., Sherman P., Low D. E., Penner J. L. Typing of Campylobacter pylori by bacterial DNA restriction endonuclease analysis and determination of plasmid profile. J Clin Microbiol. 1990 Jan;28(1):83–86. doi: 10.1128/jcm.28.1.83-86.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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