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Updated emm-typing protocol for Streptococcus pyogenes

H R Frost1, M R Davies2, S Velusamy3

  • 1Molecular Bacteriology Laboratory, Université Libre de Bruxelles, Brussels, Belgium; Tropical Diseases Research Group, Murdoch Children's Research Institute, Melbourne, Australia.

Clinical Microbiology and Infection : the Official Publication of the European Society of Clinical Microbiology and Infectious Diseases
|March 3, 2020
PubMed
Abstract

Insights

A new primer, CDC3, improves emm gene typing for Streptococcus pyogenes. This reduces misclassification of strains by ensuring PCR reactions amplify only the target emm gene sequence.

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Streptococcus pyogenes emm gene typing via PCR can lead to misclassification due to multiple band amplification.
  • Non-emm gene sequences have been mistakenly identified as emm types.

Purpose of the Study:

  • To enhance the specificity of emm typing PCR for Streptococcus pyogenes.
  • To introduce and validate a new primer, CDC3, for improved emm gene detection.

Main Methods:

  • In silico validation of the CDC3 primer against 1688 GAS genomes.
  • In vitro testing of CDC3 with 32 Streptococcus pyogenes isolates using PCR.
  • Comparison of PCR results using CDC1+CDC2 versus CDC1+CDC3 primer pairs, followed by gel electrophoresis and sequencing.

Main Results:

  • The CDC3 primer demonstrated high specificity, annealing only to emm genes in silico, unlike the CDC2 primer which annealed to non-emm genes.
  • PCR using the CDC1+CDC3 primer pair produced a single, correct-sized band in all tested isolates.
  • The CDC1+CDC2 primer pair produced multiple bands in 40% of isolates, indicating potential misclassification.

Conclusions:

  • The CDC3 primer offers superior specificity for emm gene detection in Streptococcus pyogenes compared to the CDC2 primer.
  • This new primer provides a straightforward method to prevent the mistyping of Streptococcus pyogenes strains.
Keywords:
Group A StreptococcusM proteinM-like proteinPCRStreptococcus pyogeneVaccineemm-typing protocol

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