Protocol

Diagnostic Bacteriology

Volume 1616 of the series Methods in Molecular Biology pp 71-88

Date:

Detection of Helicobacter pylori DNA in Formalin-Fixed Paraffin-Embedded Gastric Biopsies Using Laser Microdissection and qPCR

  • María Fernanda Loayza VillaAffiliated withInstituto de Microbiologia, Universidad San Francisco de QuitoUniversidad de las Fuerzas Armadas ESPE
  • , Valeria Liliana Herrera SevillaAffiliated withUniversidad de las Fuerzas Armadas ESPE
  • , Nicolás Vivar-DiazAffiliated withLaboratorios NETLAB S.A. Email author 

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Abstract

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.

Key words

Helicobacter pylori Laser microdissection qPCR detection Molecular microbiome analysis Molecular pathology