RESUMO
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 1-3 days 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 4 hours, followed by the incubation in a non-enzymatic solution with urea for 2 hours. SmaIDNA macrorestriction fragments were well resolved in 5 hours and 14 minutes by electrophoresis in a CHEF minichamber at 10 V/cm. This procedure for GAS DNA preparation was useful for fingerprinting GAS strains in the format of CHEF Mapper (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.
Assuntos
DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado/economia , Eletroforese em Gel de Campo Pulsado/métodos , Ácidos Nucleicos Imobilizados/genética , Tipagem Molecular/economia , Tipagem Molecular/métodos , Streptococcus pyogenes/classificação , Custos e Análise de Custo , DNA Bacteriano/isolamento & purificação , Humanos , Ácidos Nucleicos Imobilizados/isolamento & purificação , Epidemiologia Molecular/métodos , Streptococcus pyogenes/genética , Fatores de TempoRESUMO
The identification of specific microRNAs (miRNAs) that target a given messenger RNA (mRNA) is essential for studies in gene regulation, but the available bioinformatic software programs are often unreliable. We have developed a unique experimental miRNA affinity assay whereby a 3'UTR RNA is end-labeled with biotin, immobilized, and then used as a bait sequence for affinity pull-down of miRNAs. After washes and release, cloning and sequencing identify the miRNAs. Binding affinity is quantitated by quantitative polymerase chain reaction (qPCR), comparing released and original input concentrations. As an initial demonstration, the TCF8/ZEB1 mRNA affinity pull-down yielded miR-200 family member miRs in the majority of clones, and binding affinity was approximately 100%; virtually all copies of miR-200c bound the immobilized mRNA transcript. For validation in cells, miR-200c strongly inhibited expression of a TCF8 luciferase reporter, native TCF8 mRNA, and protein levels, which contrasted with other recovered miRNAs with lower binding affinities. For Smad4 mRNA, miR-150 (and others) displayed a binding affinity of 39% (or less) yet did not inhibit a Smad4 reporter, native Smad4 mRNA, or protein levels. These results were not predicted by available software. This work demonstrates this miRNA binding affinity assay to be a novel yet facile experimental means of identification of miRNAs targeting a given mRNA.
