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. 1992 Sep;30(9):2391–2397. doi: 10.1128/jcm.30.9.2391-2397.1992

M protein gene typing of Streptococcus pyogenes by nonradioactively labeled oligonucleotide probes.

A Kaufhold 1, A Podbielski 1, D R Johnson 1, E L Kaplan 1, R Lütticken 1
PMCID: PMC265511  PMID: 1401004

Abstract

A new approach for the typing of Streptococcus pyogenes is described. Oligonucleotide probes of 30 nucleotides in length were derived from currently known sequences of the N-terminal regions of M protein genes (emm genes). The oligonucleotides were labeled with digoxigenin-dUTP and hybridized to dot-blotted genomic DNA from 116 group A streptococcal strains of serotypes M-1, M-2, M-3, M-5, M-6, M-12, M-18, M-19, M-24, and M-49. Hybridization reactions were visualized with a chemiluminescent substrate. In comparison with conventional serological typing of expressed M proteins, the binding of the probes to the corresponding emm genes exhibited 100% sensitivity and specificity. The results emphasize the high degree of type-specific conservation of the N-terminal regions of emm genes from reference strains and epidemiologically unrelated U.S. and European clinical isolates. The existence of two distinct genetic subgroups among eight investigated M-49 strains was unequivocally shown by hybridization assays and further confirmed by nucleotide sequence data obtained from four selected M-49 strains. Because oligonucleotide probes are relatively easy to prepare, easy to handle, and known to give consistent interlaboratory results, the "oligotyping" technique appears to offer potential advantages over conventional serological typing methods.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bisno A. L. Group A streptococcal infections and acute rheumatic fever. N Engl J Med. 1991 Sep 12;325(11):783–793. doi: 10.1056/NEJM199109123251106. [DOI] [PubMed] [Google Scholar]
  2. Bollet C., Gevaudan M. J., de Lamballerie X., Zandotti C., de Micco P. A simple method for the isolation of chromosomal DNA from gram positive or acid-fast bacteria. Nucleic Acids Res. 1991 Apr 25;19(8):1955–1955. doi: 10.1093/nar/19.8.1955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cleary P. P., Kaplan E. L., Livdahl C., Skjold S. DNA fingerprints of Streptococcus pyogenes are M type specific. J Infect Dis. 1988 Dec;158(6):1317–1323. doi: 10.1093/infdis/158.6.1317. [DOI] [PubMed] [Google Scholar]
  4. Fischetti V. A. Streptococcal M protein: molecular design and biological behavior. Clin Microbiol Rev. 1989 Jul;2(3):285–314. doi: 10.1128/cmr.2.3.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. GOODER H. Association of a serum opacity reaction with serological type in Streptococcus pyogenes. J Gen Microbiol. 1961 Jul;25:347–352. doi: 10.1099/00221287-25-3-347. [DOI] [PubMed] [Google Scholar]
  6. Haanes-Fritz E., Kraus W., Burdett V., Dale J. B., Beachey E. H., Cleary P. Comparison of the leader sequences of four group A streptococcal M protein genes. Nucleic Acids Res. 1988 May 25;16(10):4667–4677. doi: 10.1093/nar/16.10.4667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Haanes E. J., Cleary P. P. Identification of a divergent M protein gene and an M protein-related gene family in Streptococcus pyogenes serotype 49. J Bacteriol. 1989 Dec;171(12):6397–6408. doi: 10.1128/jb.171.12.6397-6408.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hollingshead S. K., Fischetti V. A., Scott J. R. Complete nucleotide sequence of type 6 M protein of the group A Streptococcus. Repetitive structure and membrane anchor. J Biol Chem. 1986 Feb 5;261(4):1677–1686. [PubMed] [Google Scholar]
  9. Huang T. T., Malke H., Ferretti J. J. Heterogeneity of the streptokinase gene in group A streptococci. Infect Immun. 1989 Feb;57(2):502–506. doi: 10.1128/iai.57.2.502-506.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Johnson D. R., Kaplan E. L. Microtechnique for serum opacity factor characterization of group A streptococci adaptable to the use of human sera. J Clin Microbiol. 1988 Oct;26(10):2025–2030. doi: 10.1128/jcm.26.10.2025-2030.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Khandke K. M., Fairwell T., Acharya A. S., Trus B. L., Manjula B. N. Complete amino acid sequence of streptococcal PepM49 protein, a nephritis-associated serotype. Conserved conformational design among sequentially distinct M protein serotypes. J Biol Chem. 1988 Apr 15;263(11):5075–5082. [PubMed] [Google Scholar]
  12. Manjula B. N., Khandke K. M., Fairwell T., Relf W. A., Sriprakash K. S. Heptad motifs within the distal subdomain of the coiled-coil rod region of M protein from rheumatic fever and nephritis associated serotypes of group A streptococci are distinct from each other: nucleotide sequence of the M57 gene and relation of the deduced amino acid sequence to other M proteins. J Protein Chem. 1991 Aug;10(4):369–384. doi: 10.1007/BF01025251. [DOI] [PubMed] [Google Scholar]
  13. Miller L., Gray L., Beachey E., Kehoe M. Antigenic variation among group A streptococcal M proteins. Nucleotide sequence of the serotype 5 M protein gene and its relationship with genes encoding types 6 and 24 M proteins. J Biol Chem. 1988 Apr 25;263(12):5668–5673. [PubMed] [Google Scholar]
  14. Mouw A. R., Beachey E. H., Burdett V. Molecular evolution of streptococcal M protein: cloning and nucleotide sequence of the type 24 M protein gene and relation to other genes of Streptococcus pyogenes. J Bacteriol. 1988 Feb;170(2):676–684. doi: 10.1128/jb.170.2.676-684.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Musser J. M., Hauser A. R., Kim M. H., Schlievert P. M., Nelson K., Selander R. K. Streptococcus pyogenes causing toxic-shock-like syndrome and other invasive diseases: clonal diversity and pyrogenic exotoxin expression. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2668–2672. doi: 10.1073/pnas.88.7.2668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Podbielski A., Kühnemund O., Lütticken R. Identification of group A type 1 streptococcal M protein gene by a non-radioactive oligonucleotide detection method. Med Microbiol Immunol. 1990;179(5):255–262. doi: 10.1007/BF00192463. [DOI] [PubMed] [Google Scholar]
  17. Podbielski A., Melzer B., Lütticken R. Application of the polymerase chain reaction to study the M protein(-like) gene family in beta-hemolytic streptococci. Med Microbiol Immunol. 1991;180(4):213–227. doi: 10.1007/BF00215250. [DOI] [PubMed] [Google Scholar]
  18. Robbins J. C., Spanier J. G., Jones S. J., Simpson W. J., Cleary P. P. Streptococcus pyogenes type 12 M protein gene regulation by upstream sequences. J Bacteriol. 1987 Dec;169(12):5633–5640. doi: 10.1128/jb.169.12.5633-5640.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rotta J., Krause R. M., Lancefield R. C., Everly W., Lackland H. New approaches for the laboratory recognition of M types of group A streptococci. J Exp Med. 1971 Nov 1;134(5):1298–1315. doi: 10.1084/jem.134.5.1298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Saiki R. K., Walsh P. S., Levenson C. H., Erlich H. A. Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6230–6234. doi: 10.1073/pnas.86.16.6230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Skjold S. A., Wannamaker L. W. Method for phage typing group A type 49 streptococci. J Clin Microbiol. 1976 Sep;4(3):232–238. doi: 10.1128/jcm.4.3.232-238.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tagg J. R., Bannister L. V. "Fingerprinting" beta-haemolytic streptococci by their production of and sensitivity to bacteriocine-like inhibitors. J Med Microbiol. 1979 Nov;12(4):397–411. doi: 10.1099/00222615-12-4-397. [DOI] [PubMed] [Google Scholar]
  23. Top F. H., Jr, Wannamaker L. W. The serum opacity reaction of Streptococcus pyogenes. The demonstration of multiple, strain-specific lipoproteinase antigens. J Exp Med. 1968 May 1;127(5):1013–1034. doi: 10.1084/jem.127.5.1013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Top F. H., Jr, Wannamaker L. W. The serum opacity reaction of Streptococcus pyogenes: frequency of production of streptococcal lipoproteinase by strains of different serological types and the relationship of M protein production. J Hyg (Lond) 1968 Mar;66(1):49–58. doi: 10.1017/s0022172400040936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Widdowson J. P., Maxted W. R., Grant D. L. The production of opacity in serum by group A streptococci and its relationship withthe presence of M antigen. J Gen Microbiol. 1970 Jun;61(3):343–353. doi: 10.1099/00221287-61-3-343. [DOI] [PubMed] [Google Scholar]

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