However, these methodologies lack specificity and can introduce bias due to over- or underestimation of the microorganisms studied. The unambiguous identification of S. pyogenes strains is the most important criterion in the study of epidemiology, pathogenesis and also for prompt treatment of infections with S. pyogenes. Genomic fingerprinting assays using random amplified polymorphic DNA (RAPD) are excellent methodologies for differentiating and tracking specific genetic elements within a complex genome or genomes (Hadrys et al., 1992). The development of sequence buy PF-562271 characterized amplified region

(SCAR) markers as molecular probes has been used in the detection of fungi (Dauch et al., 2003), yeasts (De Clercq et al., 2003), Bacillus subtilis (Felici et al., 2008), Staphylococcus xylosus (Morot-Bizot et al., 2003) and Streptococcus mutans (Chen et al., 2007). However, so far this approach has not been adopted for detecting S. pyogenes. Hence, the main objective of the present study was to develop species-specific PCR primers for accurate and rapid detection of S. pyogenes. A differentially amplified fragment

obtained from RAPD profile has been converted into a SCAR. A pair of primers was then designed and evaluated for specificity towards accurate identification of S. pyogenes. A total of 33 S. pyogenes clinical isolates were used in this study. They were CB-839 concentration collected from pharyngitis patients at Government Rajaji Hospital, Madurai, South India. Isolates were maintained in glycerol at −80 °C and subcultured on sheep blood agar

before testing. Todd–Hewitt broth was used for routine culture. The test organisms nearly used in this study were GAS SF370, GBS (ATCC27956), GCS (ATCC12394), GGS (ATCC9542), B. subtilis (ATCC11774), Staphylococcus aureus (ATCC11632), Escherichia coli (ATCC10536) and Pseudomonas aeruginosa (ATCC10145). All 33 isolates used in this study were confirmed as S. pyogenes through bacteriological analysis such as β-haemolysis (on 5% sheep blood agar plate), Gram staining, the bacitracin test, PYR test, catalase test and latex agglutination test (Streptex, Remel Laboratories, UK). Along similar lines, all the throat swabs (n=270) were analysed using the above-mentioned bacteriological methods. The preparation of genomic DNA for all 33 isolates of S. pyogenes and for the test organisms were performed as described by Schlegel et al. (2003). RAPD was performed with 12-mer H2 primer 5′-CCTCCCGCCACC-3′ sequence (Seppala et al., 1994) using a standardized protocol in a thermal cycler (GeneAmp PCR system 9700, Applied Biosystems). Each reaction mixture (25 μL total volume) contained 1 × PCR buffer [10 mM Tris-HCl (pH 8.8), 50 mM KCl], 0.2 mM dNTPs, 1.5 mM MgCl2, 50 pM of primer, 1 U of Taq polymerase (MBI Fermentas, Germany) and 10 ng of DNA as template.