3a). No amplification signal was observed at the lowest concentration of 10−3 ng−1 PCR. To estimate the sensitivity of S. pyogenes detection, serial dilutions of S. pyogenes cells were prepared in saline and 5-μL
aliquots from each dilution series were added directly to the PCR mixture containing primer pair 212F/212R for PCR reaction. Simultaneously, the aliquots (100 μL) were plated on tryptose MI-503 cost agar plates up to 10−6 dilution. A PCR amplicon of 212 bp was observed up to 10−5 dilution (Fig. 3b). The numbers of CFU observed for 100-μL aliquots of different dilutions are presented in Supporting Information, Table S1. The specificity of SCAR primers 212F/212R was evaluated against the DNA extracted from 270 clinical throat swabs of pharyngitis patients. Twenty-three samples were positive for the SCAR primers, which indicated the presence of S. pyogenes in these throat swabs. In contrast to this, only eight samples were found to be positive Metformin for S. pyogenes in the standard
bacteriological analysis. Hence the SCAR primers are found to be an efficient tool in the identification of S. pyogenes from the throat metagenome. It is important to identify S. pyogenes accurately from clinical samples as this bacterium remains a significant human pathogen among Gram-positive organisms and is responsible for a wide array of infections. For the past two decades several methods have been tried for the identification of S. pyogenes, such as the DAI test (Venezia et al., 1985), fluorescent antibody (Facklam & Carey, 1985), latex agglutination test (Gerber et al., 1984), Baricitinib enzyme immunoassay (Schwabe et al., 1991), rapid optical immunoassay technique (Harbeck et al., 1993) and DNA probe (Steed et al., 1993). Due to lack of sensitivity and
specificity, these methods are no longer used, and clinicians have switched over to molecular tools such as ribotyping (Bruneau et al., 1994; Shundi et al., 2000), RFLP analysis (Cleary et al., 1988) and REA (Bingen et al., 1992) for the identification of S. pyogenes. However, these methods usually need sequence determination and are not economical for clinical use. RAPD profiling is a molecular typing method that makes it possible to identify natural polymorphisms using a single, short oligonucleotide primer. This method is faster, technically less demanding and more economical (Seppala et al., 1994). In addition, this method produces a high range of profiling with a very low stringency (Wang et al., 1993). This agrees with our RAPD profile, where the 33 isolates were classified into eight groups (A–H). Profiles A, F and G were observed in 42%, 30% and 9% of isolates, respectively. The H2 primer generated two to eight bands of varying sizes and eight different strains were observed within the 33 S. pyogenes isolates.