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

  • Yaumara Lopez Cuban Neurosciences Center, La Habana, Cuba
  • Viana Manrique Cuban Neurosciences Center, La Habana, Cuba
  • Esperanza Niubo Cuban Neurosciences Center, La Habana, Cuba
  • Margarita Valdés-Dapena Pediatric Hospital Juan M. Márquez, La Habana, Cuba
  • Julian Perez Amarillo Pediatric Hospital Juan M. Márquez, La Habana, Cuba
  • Israel Lopez Autonomous University of Mexico City (UACM), México DF, México
  • Berenice Parra CINVESTAV-IPN, México DF, México
  • Gerardo Escalona Venegas Pediatric Hospital of Mexico Federico Gómez, México DF, México
  • Ana M Riveron Cuban Neurosciences Center, La Habana, Cuba
  • Lilia Lopez-Canovas Cuban Neurosciences Center, La Habana, Cuba
Keywords: GAS DNA, miniPFGE, bacterial subtyping, non-enzymatic methods

Abstract

Introduction: Group A streptococci (GAS) is responsible of several human diseases ranging from mild infection to severe invasive toxin-mediated 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.

Author Biographies

Yaumara Lopez, Cuban Neurosciences Center, La Habana, Cuba

Molecular Biology Department

Doctoral Student

Viana Manrique, Cuban Neurosciences Center, La Habana, Cuba

Molecular Biology Department

Doctoral Student

Esperanza Niubo, Cuban Neurosciences Center, La Habana, Cuba

Molecular Biology Department

Research Associate

Margarita Valdés-Dapena, Pediatric Hospital Juan M. Márquez, La Habana, Cuba

Clinical Microbiology Laboratory

Senior Researcher

Julian Perez Amarillo, Pediatric Hospital Juan M. Márquez, La Habana, Cuba

Clinical Microbiology Laboratory

Senior Researcher

Israel Lopez, Autonomous University of Mexico City (UACM), México DF, México

Academy of Sciencies and Humanities

Research Professor

Berenice Parra, CINVESTAV-IPN, México DF, México

Cell Biology Department

Post-Doctoral Student

Gerardo Escalona Venegas, Pediatric Hospital of Mexico Federico Gómez, México DF, México

Laboratory of Intestinal Bacteriology

Doctoral Student

Ana M Riveron, Cuban Neurosciences Center, La Habana, Cuba

Molecular Biology Department

Senior Researcher

Lilia Lopez-Canovas, Cuban Neurosciences Center, La Habana, Cuba

Postgraduate Program in Genomic Sciences

Research Professor

Published
2015-07-30
How to Cite
1.
Lopez Y, Manrique V, Niubo E, Valdés-Dapena M, Perez Amarillo J, Lopez I, Parra B, Escalona Venegas G, Riveron AM, Lopez-Canovas L (2015) Cost-effective procedure for Streptococcus pyogenes immobilized DNA preparation and miniPFGE subtyping. J Infect Dev Ctries 9:710-719. doi: 10.3855/jidc.6115
Section
Original Articles