1887

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Volume 55, Issue 8

Genotyping of using seven single-nucleotide polymorphisms in combination with short variable region sequencing Free

Abstract

This investigation describes the development of a generally applicable, bioinformatics-driven, single-nucleotide polymorphism (SNP) genotyping assay for the common bacterial gastrointestinal pathogen . SNPs were identified using the program ‘Minimum SNPs’, which selects for polymorphisms providing the greatest resolution of bacterial populations based on Simpson's index of diversity (). The high- SNPs identified in this study were derived from the combined / multilocus sequence typing (MLST) database. Seven SNPs were found that provided a of 0.98 compared with full MLST characterization, based on 959 sequence types (STs). The seven high- SNPs were interrogated using allele-specific real-time PCR (AS kinetic PCR), which negates the need for expensive labelled primers or probes and requires minimal assay optimization. The total turnaround time of the SNP typing assay was approximately 2 h. Concurrently, 69 isolates were subjected to MLST and flagellin A short variable region ( SVR) sequencing and combined with a population of 84 and isolates previously characterized by these methods. Within this collection of 153 isolates, 19 SVR types (=0.857) were identified, compared with 40 different STs (=0.939). When MLST and SVR sequencing were used in combination, the discriminatory power was increased to 0.959. In comparison, SNP typing of the 153 isolates alone provided a of 0.920 and was unable to resolve a small number of unrelated isolates. However, addition of the SVR locus to the SNP typing procedure increased the resolving power to 0.952 and clustered isolates similarly to MLST/ SVR. This investigation has shown that a seven-member SNP typing assay, used in combination with sequencing of the SVR, efficiently discriminates isolates.

  • Received:
  • Accepted:
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Keyword(s): AS kinetic PCR, allele-specific real-time PCR , AS, allele-specific , CC, clonal complex , D, Simpson's index of diversity , flaA SVR, flagellin A short variable region , gDNA, genomic DNA , MLST, multilocus sequence typing , SLV, single-locus variant , SNP, single-nucleotide polymorphism , ST, sequence type and Tm, melting temperature
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2006-08-01
2024-04-27
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References

  1. Altekruse S. F., Stern N. J., Fields P. I., Swerdlow D. L. 1999; Campylobacter jejuni – an emerging foodborne pathogen. Emerg Infect Dis 5:28–35 [CrossRef]
     [Google Scholar]
  2. Best E. L., Fox A. J., Frost J. A., Bolton F. J. 2004; Identification of Campylobacter jejuni multilocus sequence type ST-21 clonal complex by single-nucleotide polymorphism analysis. J Clin Microbiol 42:2836–2839 [CrossRef]
     [Google Scholar]
  3. Best E. L., Fox A. J., Frost J. A., Bolton F. J. 2005; Real-time single-nucleotide polymorphism profiling using Taqman technology for rapid recognition of Campylobacter jejuni clonal complexes. J Med Microbiol 54:919–925 [CrossRef]
     [Google Scholar]
  4. Clark C. G., Bryden L., Cuff W. R., Johnson P. L., Jamieson F., Ciebin B., Wang G. 2005; Use of the Oxford multilocus sequence typing protocol and sequencing of the flagellin short variable region to characterise isolates from a large outbreak of waterborne Campylobacter sp. strains in Walkerton, Ontario, Canada. J Clin Microbiol 43:2080–2091 [CrossRef]
     [Google Scholar]
  5. Dingle K. E., Colles F. M., Wareing D. R. & 7 other authors (2001a). Multilocus sequence typing system for Campylobacter jejuni . J Clin Microbiol 39:14–23 [CrossRef]
     [Google Scholar]
  6. Dingle K. E., Van Den Braak N., Colles F. M., Price L. J., Woodward D. L., Rodgers F. G., Endtz H. P., van Belkum A., Maiden M. C. 2001b; Sequence typing confirms that Campylobacter jejuni strains associated with Guillain–Barré and Miller–Fisher syndromes are of diverse genetic lineage, serotype, and flagella type. J Clin Microbiol 39:3346–3349 [CrossRef]
     [Google Scholar]
  7. Dingle K. E., Colles F. M., Ure R., Wagenaar J. A., Duim B., Bolton F. J., Fox A. J., Wareing D. R., Maiden M. C. 2002; Molecular characterization of Campylobacter jejuni clones: a basis for epidemiologic investigation. Emerg Infect Dis 8:949–955 [CrossRef]
     [Google Scholar]
  8. Dingle K. E., Colles F. M., Falush D., Maiden M. C. 2005; Sequence typing and comparison of population biology of Campylobacter coli and Campylobacter jejuni . J Clin Microbiol 43:340–347 [CrossRef]
     [Google Scholar]
  9. Duim B., Godschalk P. C., van den Braak N. & 9 other authors; 2003; Molecular evidence for dissemination of unique Campylobacter jejuni clones in Curaçao, Netherlands Antilles. J Clin Microbiol 41:5593–5597 [CrossRef]
     [Google Scholar]
  10. Feil E. J., Cooper J. E., Grundmann H. & 9 other authors; 2003; How clonal is Staphylococcus aureus ?. J Bacteriol 185:3307–3316 [CrossRef]
     [Google Scholar]
  11. Feil E. J., Li B. C., Aanensen D. M., Hanage W. P., Spratt B. G. 2004; eburst: inferring patterns of evolutionary descent among clusters of related bacterial genotypes from multilocus sequence typing data. J Bacteriol 186:1518–1530 [CrossRef]
     [Google Scholar]
  12. French N., Barrigas M., Brown P. & 7 other authors; 2005; Spatial epidemiology and natural population structure of Campylobacter jejuni colonizing a farmland ecosystem. Environ Microbiol 7:1116–1126 [CrossRef]
     [Google Scholar]
  13. Germer S., Higuchi R. 1999; Single-tube genotyping without oligonucleotide probes. Genome Res 9:72–78
     [Google Scholar]
  14. Germer S., Higuchi R. 2003; Homogeneous allele-specific PCR in SNP genotyping. Methods Mol Biol 212:197–214
     [Google Scholar]
  15. Heid C. A., Stevens J., Livak K. J., Williams P. M. 1996; Real time quantitative PCR. Genome Res 6:986–994 [CrossRef]
     [Google Scholar]
  16. Hézard N., Cornillet P., Droullé C., Gillot L., Potron G., Nguyen P. 1997; Factor V Leiden: detection in whole blood by ASA PCR using an additional mismatch in antepenultimate position. Thromb Res 88:59–66 [CrossRef]
     [Google Scholar]
  17. Hunter P. R., Gaston M. A. 1988; Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J Clin Microbiol 26:2465–2466
     [Google Scholar]
  18. Huygens F., Stephens A. J., Nimmo G. R., Giffard P. M. 2004; mecA locus diversity in methicillin-resistant Staphylococcus aureus isolates in Brisbane, Australia, and the development of a novel diagnostic procedure for the Western Samoan phage pattern clone. J Clin Microbiol 42:1947–1955 [CrossRef]
     [Google Scholar]
  19. Kapperud G., Espeland G., Wahl E. & 7 other authors; 2003; Factors associated with increased and decreased risk of Campylobacter infection: a prospective case-control study in Norway. Am J Epidemiol 158:234–242 [CrossRef]
     [Google Scholar]
  20. Livak K. J. 1999; Allelic discrimination using fluorogenic probes and the 5′ nuclease assay. Genet Anal 14:143–149 [CrossRef]
     [Google Scholar]
  21. Maiden M. C., Bygraves J. A., Feil E. & 10 other authors; 1998; Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A 95:3140–3145 [CrossRef]
     [Google Scholar]
  22. Meinersmann R. J., Helsel L. O., Fields P. I., Hiett K. L. 1997; Discrimination of Campylobacter jejuni isolates by fla gene sequencing. J Clin Microbiol 35:2810–2814
     [Google Scholar]
  23. Mellmann A., Mosters J., Bartelt E., Roggentin P., Ammon A., Friedrich A. W., Karch H., Harmsen D. 2004; Sequence-based typing of flaB is a more stable screening tool than typing of flaA for monitoring of Campylobacter populations. J Clin Microbiol 42:4840–4842 [CrossRef]
     [Google Scholar]
  24. Mhlanga M. M., Malmberg L. 2001; Using molecular beacons to detect single-nucleotide polymorphisms with real-time PCR. Methods 25:463–471 [CrossRef]
     [Google Scholar]
  25. Miller W. G., On S. L. W., Wang G., Fontanoz S., Lastovica A. J., Mandrell R. E. 2005; Extended multilocus sequence typing system for Campylobacter coli , C. lari , C. upsaliensis , and C. helveticus . J Clin Microbiol 43:2315–2329 [CrossRef]
     [Google Scholar]
  26. Nachamkin I., Bohachick K., Patton C. M. 1993; Flagellin gene typing of Campylobacter jejuni by restriction fragment length polymorphism analysis. J Clin Microbiol 31:1531–1536
     [Google Scholar]
  27. Newton C. R., Graham A., Heptinstall L. E., Powell S. J., Summers C., Kalsheker N., Smith J. C., Markham A. F. 1989; Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 17:2503–2516 [CrossRef]
     [Google Scholar]
  28. O'Reilly L. C., Inglis T. J., Unicomb L. the Australian Campylobacter Subtyping Study Group (2006). Australian multicentre comparison of subtyping methods for the investigation of Campylobacter infection. Epidemiol Infect (epub ahead of print)
    [Google Scholar]
  29. Papp A. C., Pinsonneault J. K., Cooke G., Sadee W. 2003; Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves. Biotechniques 34:1068–1072
     [Google Scholar]
  30. Pebody R. G., Ryan M. J., Wall P. G. 1997; Outbreaks of campylobacter infection: rare events for a common pathogen. Commun Dis Rep CDR Rev 7:R33–R37
     [Google Scholar]
  31. Robertson G. A., Thiruvenkataswamy V., Shilling H., Price E. P., Huygens F., Henskens F. A., Giffard P. M. 2004; Identification and interrogation of highly informative single nucleotide polymorphism sets defined by bacterial multilocus sequence typing databases. J Med Microbiol 53:35–45 [CrossRef]
     [Google Scholar]
  32. Sails A. D., Swaminathan B., Fields P. I. 2003; Utility of multilocus sequence typing as an epidemiological tool for investigation of outbreaks of gastroenteritis caused by Campylobacter jejuni . J Clin Microbiol 41:4733–4739 [CrossRef]
     [Google Scholar]
  33. Schouls L. M., Reulen S., Duim B., Wagenaar J. A., Willems R. J., Dingle K. E., Colles F. M., Van Embden J. D. 2003; Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism, multilocus sequence typing, and short repeat sequencing: strain diversity, host range, and recombination. J Clin Microbiol 41:15–26 [CrossRef]
     [Google Scholar]
  34. Shi M. M. 2001; Enabling large-scale pharmacogenetic studies by high-throughput mutation detection and genotyping technologies. Clin Chem 47:164–172
     [Google Scholar]
  35. Spratt B. G., Hanage W. P., Li B., Aanensen D. M., Feil E. J. 2004; Displaying the relatedness among isolates of bacterial species – the eburst approach. FEMS Microbiol Lett 241:129–134 [CrossRef]
     [Google Scholar]
  36. Stephens A. J., Huygens F., Inman-Bamber J., Price E. P., Nimmo G. R., Schooneveldt J., Munckhof W., Giffard P. M. 2006; Methicillin resistant Staphylococcus aureus genotyping using a small set of polymorphisms. J Med Microbiol 55:43–51 [CrossRef]
     [Google Scholar]
  37. Suerbaum S., Lohrengel M., Sonnevend A., Ruberg F., Kist M. 2001; Allelic diversity and recombination in Campylobacter jejuni . J Bacteriol 183:2553–2559 [CrossRef]
     [Google Scholar]
  38. van Belkum A. 2003; High-throughput epidemiologic typing in clinical microbiology. Clin Microbiol Infect 9:86–100 [CrossRef]
     [Google Scholar]
  39. Wassenaar T. M., Fry B. N., van der Zeijst B. A. 1995; Variation of the flagellin gene locus of Campylobacter jejuni by recombination and horizontal gene transfer. Microbiology 141:95–101 [CrossRef]
     [Google Scholar]
  40. Wu D. Y., Ugozzoli L., Pal B. K., Wallace R. B. 1989; Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci U S A 86:2757–2760 [CrossRef]
     [Google Scholar]
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Genotyping of Campylobacter jejuni using seven single-nucleotide polymorphisms in combination with flaA short variable region sequencing
J. Med. Microbiol. 55, 1061 (2006); https://doi.org/10.1099/jmm.0.46460-0
/content/journal/jmm/10.1099/jmm.0.46460-0
/content/journal/jmm/10.1099/jmm.0.46460-0
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