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

摘要

Graphical abstract Display Omitted Abstract 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 10–20μm of diameter and 3μm thick with more than 10 bacteria in each cut. ? Lysis with 30μL of tissue lysis buffer and 20μL of proteinase K (PK) with the tube in an upside-down position. ? The use of thin purification columns with 35μL of elution buffer. The mean of DNA concentration obtained from 25 LM cut sections was 1.94±0.16ng/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.