RESUMO

Molecular detection and analysis of virulence factors of Helicobacter pylori depends on the specificity of cell selection in the gastric biopsies. The laser microdissection (LM) instruments combine microscopy with laser cut sectioning. This combination allows one to choose only the bacteria that are in direct contact with epithelial cells in the gastric biopsy sample, avoiding those microorganisms attached to the mucus layer in the sample. The average concentration of DNA isolated from 25 cuts with selected bacteria is around 1.94 ng/µL, which is enough DNA to perform a qPCR protocol using real-time instruments to amplify 16sDNA or virulence factors like cagA or vacA. Consequently, the application of these technologies in the molecular analysis of Helicobacter pylori directly in contact with the surface of gastric epithelial cells is more precise and could yield better insights about the complex mechanisms of interactions between pathogen and host. Insights derived from research using the techniques described herein may in future facilitate prevention of infection or improved therapeutic options.

Assuntos

Mucosa Gástrica/patologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Microdissecção e Captura a Laser/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , DNA Bacteriano/isolamento & purificação , Formaldeído , Mucosa Gástrica/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/isolamento & purificação , Humanos , Inclusão em Parafina , Fixação de Tecidos/métodos

RESUMO

To assess the molecular events exerted by Helicobacter pylori interacting directly with gastric epithelial cells, an improved procedure for microbial DNA isolation from stained hematoxilin-eosin gastric biopsies was developed based on laser micro-dissection (LM) [1]. Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue [2]. To improve the yield and quality of DNA isolated from H. pylori contacting intestinal epithelial cells, the following conditions were established after modification of the QIAamp DNA Micro kit. •Use of at least 25 cut sections of 10-20 µm of diameter and 3 µm thick with more than 10 bacteria in each cut.•Lysis with 30 µL of tissue lysis buffer and 20 µL of proteinase K (PK) with the tube in an upside-down position.•The use of thin purification columns with 35 µL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94± 0 .16 ng/µL, and it was efficiently amplified with qPCR in a Bio Rad iCycler instrument. The LM can improve the sample selection and DNA extraction for molecular analysis of H. pylori associated with human gastric epithelium.