Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection

摘要

class="kwd-title">Keywords: Laser micro-dissection, H. pylori detection, FFPE tissue, DNA extraction, Gastric biopsie class="head no_bottom_margin" id="idm140381902222384title">AbstractTo 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) . Few articles have described the use of LM to select and detect H. pylori genome from formalin-fixed paraffin embedded gastric tissue . 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. class="first-line-outdent">

• 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.