Updated emm-typing protocol for Streptococcus pyogenes

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.