Development of this marker circumvents the need for culturing methods before analysis. As they are more species-specific, they target

a known sequence and detect with a single band instead of a profile (Pujol et al., 2005). In the present study, the MB of 419 bp was observed exclusively in S. pyogenes strains. The unambiguous identification of the S. pyogenes strain-specific band led to the designing of SCAR primers within the MB fragment. The MB fragment codes for the enzyme 3-keto acyl reductase (FabG), which plays a key role in lipid biosynthesis. FabG is a member of the keto acyl reductase family of proteins and is an essential enzyme for type II fatty acid biosynthesis (Lai & Cronan, 2004). Selleck Nutlin-3a Even though this enzyme is common in all bacterial organisms, the multiple-sequence alignment screening of amino acid sequences of this MB fragment showed <90% similarity with other species of Streptococcus. Hence this MB fragment is useful in the design of a species-specific marker against S. pyogenes. Consequently, a primer pair was designed within the internal region of MB with a resulting product size of 212 bp (SCAR), which is unique for S. pyogenes. Further, the specificity of SCAR primers was confirmed by the amplified product of 212 bp

in all S. pyogenes strains with the absence of nonspecific amplification signals. The specificity was also confirmed with other bacterial genera. The MAPK inhibitor PCR sensitivity assessed by means of serial dilutions of DNA extracted from pure cell cultures of S. pyogenes resulted in a range of about 100–10−1 ng of template (Fig. 3a). However, when the aliquots of the serially diluted oxyclozanide cell cultures were taken directly for PCR, amplification could be observed from 5-μL aliquots from 10−5 dilution. The number of CFUs observed in 100 μL of 10−5 dilution

was 32. This implies that PCR using SCAR primers is sensitive enough to detect one or two planktonic cells of S. pyogenes. These experiments substantiate the threshold level of qualitative PCR for the detection of amplification signal. The development of the SCAR primers may reduce the prevailing uncertainty in the identification of S. pyogenes. Earlier reports state that GCS and GGS express Lancefield’s group A antigen, which leads to the misidentification of S. pyogenes. GCS and GGS have traditionally been considered commensal organisms found as part of the normal flora of the skin, throat and other mucosal surfaces and therefore only caused opportunistic infections in individuals with underlying risk factors. However, GGS is increasingly associated with a spectrum of diseases in healthy individuals that overlaps that of GAS. This sharing of similar antigens between two different Streptococcus species might be due to the interspecies recombinational exchanges from GAS donors to GCS–GGS recipients.