Application of the polymerase chain reaction to study the M protein(-like) gene family in beta-hemolytic streptococci

Abstract

Evaluation of homologous regions of published M protein (emm) gene sequences from group A streptococci (GAS; Streptococcus pyogenes) was used to design three primer pairs for polymerase chain reaction (PCR) and three oligonucleotide probe sequences internal to the amplified products. One set of primers and corresponding probe should detect and lead to amplification of emm(-like) genes of virtually every type ("all M"), another ("SOR-M") should only amplify emm(-like) genes from GAS negative for serum opacity reaction (SOR) and the third ("SOR+ M") should expand only emm(-like) genes from SOR+ GAS. Using the "allM" primer pair for PCR on the genomic DNA from GAS of 29 different M types as well as from a group C and a group G streptococcal isolate, DNA fragments within the expected size range were amplified in every assay. All PCR products reacted with the "allM" probe. Related sequences were not detected in genomic DNA of an S. agalactiae and an Enterococcus faecalis isolate. Applying the "SOR-M" and "SOR+M" primers to identical assays led to mutually exclusive amplification products. The "SOR-M" and "SOR+M" probes hybridized only to their corresponding products. Exceptions to this exclusivity were the SOR- GAS of M types 3, 8, 27, 34, 42, 67, and 69, which consistently reacted only with the "SOR+M" primer/probe set. Analysis of sequence data from the amplified emm(-like) 2, 3, 18, and 19 genes revealed interesting specific features such as conserved gaps in the C-terminal sequence regions from SOR+ and the exceptional SOR- GAS strains. These data indicate the existence of a subgroup of strains among SOR- GAS and may advance our understanding of phylogenetic relationship between different serotypes of GAS.