title: Improved method for extraction and detection of Helicobacter pylori DNA in formalin-fixed paraffin embedded gastric biopsies using laser micro-dissection.
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 10-20 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.
authors.affiliation: Universidad de las Fuerzas Armadas ESPE, P.O. Box 171-5-231B, Av. General Rumiñahui s/n, Sangolquí, Ecuador ; Hospital Carlos Andrade Marín, P.O. Box 170411, Av. 18 de Septiembre s/n y Ayacucho, Quito, Ecuador.
authors.forename: María Fernanda
authors.initials: MF
authors.lastname: Loayza
authors.affiliation: Universidad de las Fuerzas Armadas ESPE, P.O. Box 171-5-231B, Av. General Rumiñahui s/n, Sangolquí, Ecuador.
authors.forename: Fernando Xavier
authors.initials: FX
authors.lastname: Villavicencio
authors.affiliation: Centro de Investigación Traslacional, Universidad de las Américas, Calle José Queri. Quito, Ecuador.
authors.forename: Stephanie Carolina
authors.initials: SC
authors.lastname: Santander
authors.affiliation: Centro de Investigación Traslacional, Universidad de las Américas, Calle José Queri. Quito, Ecuador.
authors.forename: Manuel
authors.initials: M
authors.lastname: Baldeón
authors.affiliation: Universidad de las Fuerzas Armadas ESPE, P.O. Box 171-5-231B, Av. General Rumiñahui s/n, Sangolquí, Ecuador.
authors.forename: Lourdes Karina
authors.initials: LK
authors.lastname: Ponce
authors.affiliation: Hospital Carlos Andrade Marín, P.O. Box 170411, Av. 18 de Septiembre s/n y Ayacucho, Quito, Ecuador.
authors.forename: Iván
authors.initials: I
authors.lastname: Salvador
authors.affiliation: Hospital Carlos Andrade Marín, P.O. Box 170411, Av. 18 de Septiembre s/n y Ayacucho, Quito, Ecuador ; NETLAB S.A., Calle "A" (Oe7A) N31-145 y Av. Mariana de Jesús, Quito, Ecuador.
authors.forename: Nicolás
authors.initials: N
authors.lastname: Vivar Díaz
keywords: DNA extraction; FFPE tissue; Gastric biopsie; H. pylori detection; Laser micro-dissection