Cost-effective procedure for Streptococcus pyogenes immobilized DNA preparation and miniPFGE subtyping

Abstract

Introduction: Group A streptococci (GAS) is responsible of several human diseases ranging from mild infection to severe invasive toxinmediated disease and post-infectious sequelae. Accordingly, a GAS surveillance program based on molecular techniques is advisable for its epidemiological control. Pulsed-field gel electrophoresis (PFGE) is the gold standard for GAS molecular subtyping, but a major disadvantage is the length of the procedure, which takes 1-3days of work, minimum. The aim of this study was to develop a rapid and cost-effective procedure for PFGE subtyping of GAS isolates. Methodology: Different incubation times of GAS, immobilized in agarose miniplugs, in solutions containing lysozyme and/or mutanolysine followed by solutions with urea instead of proteinase K, were assayed. DNA was restricted with SmaI and the fingerprints were obtained in clamped homogeneous electric field (CHEF) chambers and minichambers. The modified procedure was used to subtype 22 GAS isolates. Results: Intact DNA molecules of GAS immobilized in agarose miniplugs were prepared incubating the cells, in situ, with a solution containing lysozyme for 4hours, followed by the incubation in a non-enzymatic solution with urea for 2hours. SmaIDNA macrorestriction fragments were well resolved in 5hours and 14minutes by electrophoresis in a CHEF minichamber at 10V/cm. This procedure for GAS DNA preparation was useful for fingerprinting GAS strains in the format of CHEFMapper (BioRad). Conclusions: The procedure took 13 hours for GAS strains subtyping. Both sample preparation and electrophoresis in CHEF minichamber represent an economic alternative for performing massive epidemiological studies of this human pathogen.