Abstract
An oligonucleotide probe of 30 nucleotides length has been constructed, spanning the codons of amino acids 2 to 11 of the mature M1 protein of group A streptococci (Streptococcus pyogenes). It was labeled with digoxigenin-dUTP and visualized after hybridization with an anti-digoxigenin-antibody/alkaline phosphatase conjugate. It definitely detected the emm1 gene in dot-blotted 20-μg amounts of total nucleid acid extracts. When tested with 27 type M1 group A streptococci of several epidemiologically unrelated outbreaks and 24 non-M1 strains, its sensitivity and specificity of detection reached 100% even at a hybridization temperature 35°C below the calculated Tm. A detailed protocol for the construction and use of this oligonucleotide probe is given.
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References
Caulfield MJ, Shaffer D (1984) A computer program for the evaluation of ELISA data obtained using an automated microtiter plate absorbance reader. J Immunol Methods 74:205–215
Fischetti VA (1989) Streptococcal M protein: molecular design and biological behavior. Clin Microbiol Rev 2:285–314
Haanes-Fritz E, Kraus W, Burdett V, Dale JB, Beachey EH, Cleary P (1988) Comparison of the leader sequences of four group A streptococcal M protein genes. Nucleic Acids Res 16:4667–4677
Haanes EJ, Cleary PP (1989) Identification of a divergent M protein gene and an M proteinrelated gene family in Streptococcus pyogenes serotype 49. J Bacteriol 171:6397–6408
Hollingshead SK, Fischetti VA, Scott JR (1986) Complete nucleotide sequence of type 6 M protein of the group A streptococcus. J Biol Chem 261:1677–1686
Huang TT, Malke H, Ferretti JJ (1989) Heterogeneity of the streptokinase gene in group A streptococci. Infect Immun 57:502–506
Lancefield RC (1938) A microprecipitin technique for classifying hemolytic streptococci and improved methods for producing antisera. Proc Soc Exp Biol Med 38:473–481
Lancefield RC, Perlmann G (1952) Preparation and properties of type specific M-antigen isolated from a group A type 1 streptococcus. J Exp Med 96:71–82
Maxted WR, Widdowson JP, Fraser CAM, Ball LC, Bassett DCJ (1973) The use of the serum opacity reaction in the typing of group A streptococci. J Med Microbiol 6:83–90
Meinkoth J, Wahl G (1984) Hybridization of nucleic acids immobilized on solid supports. Anal Biochem 138:267–284
Miller L, Gray L, Beachey E, Kehoe M (1988) Antigenic variation among group A streptococci M proteins. J Biol Chem 263:5668–5673
Mouw AR, Beachey EH, Burdett V (1988) 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 170:676–684
Robbins JC, Spanier JG, Jones SJ, Simpson WJ, Cleary PP (1987) Streptococcus pyogenes type 12M protein gene regulation by upstream sequences. J Bacteriol 169:5633–5640
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, p 7–54
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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 179, 255–262 (1990). https://doi.org/10.1007/BF00192463
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DOI: https://doi.org/10.1007/BF00192463