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Identification of group A type 1 streptococcal M protein gene by a non-radioactive oligonucleotide detection method
A Podbielski1, O Kühnemund, R Lütticken
1Institute of Medical Microbiology, Klinikum der RWTH, Aachen, Federal Republic of Germany.
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-micrograms amounts of total nucleoid 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 degrees C below the calculated Tm. A detailed protocol for the construction and use of this oligonucleotide probe is given.
Insights
A new oligonucleotide probe accurately detects the emm1 gene in Streptococcus pyogenes. This highly sensitive and specific probe offers a reliable method for identifying M1 group A streptococci strains.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Group A streptococci (GAS) are significant human pathogens.
- Accurate identification of M1 serotype GAS is crucial for epidemiological surveillance and clinical management.
- Existing diagnostic methods may have limitations in sensitivity or specificity.
Purpose of the Study:
- To develop and validate a novel oligonucleotide probe for the specific detection of the emm1 gene in Streptococcus pyogenes.
- To assess the sensitivity and specificity of the probe in identifying M1 GAS strains.
Main Methods:
- Construction of a 30-nucleotide oligonucleotide probe targeting codons 2-11 of the M1 protein.
- Labeling the probe with digoxigenin-dUTP.
- Hybridization assays using dot-blotted total nucleoid acid extracts from various Streptococcus pyogenes strains.
Main Results:
- The oligonucleotide probe successfully detected the emm1 gene in 20-microgram total nucleic acid extracts.
- 100% sensitivity and specificity were achieved when testing against 27 M1 GAS strains and 24 non-M1 strains.
- Effective detection was observed even at a hybridization temperature 35°C below the calculated Tm.
Conclusions:
- The developed oligonucleotide probe provides a highly sensitive and specific tool for identifying emm1-positive Streptococcus pyogenes.
- This method offers a reliable and potentially rapid diagnostic approach for M1 GAS detection.
- The probe's performance at reduced hybridization temperatures suggests robustness and potential for optimized protocols.