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

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Diagnostic Bacteriology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1616))

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.

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Author information

Authors and Affiliations

  1. Instituto de Microbiologia, Universidad San Francisco de Quito, P.O. Box 17-1200-841, Av. Diego de Robles y Vía Interoceánica, Quito, Ecuador

    María Fernanda Loayza Villa

  2. Universidad de las Fuerzas Armadas ESPE, P.O. Box 171-5-231B, Av. General Rumiñahui s/n, Sangolquí, Ecuador

    Valeria Liliana Herrera Sevilla

  3. Laboratorios NETLAB S.A., P.O. Box 170411, Calle “A” (Oe7A) N31-145 y Mariana de Jesús, Quito, Ecuador

    Nicolás Vivar-Diaz

  4. Universidad de las Fuerzas Armadas ESPE, Sangolquí, Ecuador

    María Fernanda Loayza Villa

Authors
  1. María Fernanda Loayza Villa
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  2. Valeria Liliana Herrera Sevilla
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  3. Nicolás Vivar-Diaz
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Corresponding author

Correspondence to Nicolás Vivar-Diaz .

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Editors and Affiliations

  1. Germantown, Maryland, USA

    Kimberly A. Bishop-Lilly

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Loayza Villa, M.F., Herrera Sevilla, V.L., Vivar-Diaz, N. (2017). Detection of Helicobacter pylori DNA in Formalin-Fixed Paraffin-Embedded Gastric Biopsies Using Laser Microdissection and qPCR. In: Bishop-Lilly, K. (eds) Diagnostic Bacteriology. Methods in Molecular Biology, vol 1616. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7037-7_4

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  • DOI: https://doi.org/10.1007/978-1-4939-7037-7_4

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7035-3

  • Online ISBN: 978-1-4939-7037-7

  • eBook Packages: Springer Protocols

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