Related Experiment Video
Updated: Jul 9, 2025
Gastric Mucosa Quantitative Polymerase Chain Reaction Analysis for Detecting Helicobacter pylori and Antibiotic Resistance
Published on: March 7, 2025
An Efficient and Easy- to- Use Method for Extraction of H. pylori DNA from Archival Formalin-Fixed and
Parastoo Saniee1, Paria Ghadersoltani1, Masoumeh Noroozpour1
1Department of Microbiology and Microbial Biotechnology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University G.C, Tehran, Iran.
Background & Objective:
Formalin-fixed paraffin-embedded archived tissues are useful for the genetic analyses and assessment of some patients' disease history, including infectious diseases. However, there is no established protocol for extracting bacterial DNA from the archived specimens. In this study DNA was extracted from the archived H. pylori-positive gastric biopsies by some modifications applied to the previously published protocols. The quality of the extracted DNA was assessed by amplifying H. pylori-specific 16S rRNA gene.
Methods:
Fifty H. pylori-positive gastric biopsies obtained, fixed, and embedded in paraffin blocks during 2002-2008 were recruited. After paraffin removal, simultaneous proteinase K treatment and mechanical disruption using glass beads were used for the digestion of gastric tissues. DNA extraction was performed by adding one step of phenol treatment and two steps of incubation to the conventional phenol-chloroform method. The quantity and quality of the extracted DNA samples were assessed. Also, PCR was performed using primers specific for the H. pylori-specific 16S rRNA.
Results:
The electrophoresis showed that intact DNAs were recovered from all biopsy samples. Amplification of the PCR products with the size of 519bp confirmed the presence of H. pylori-specific 16S rRNA gene in all the biopsies.
Conclusion:
A 100% success rate for the amplification of H. pylori-specific 16S rRNA gene was achieved from all the samples. In this regard, the designed modified method resulted in the effective removal of interfering contaminations and enhanced the quality of the extracted bacterial DNA from the archived tissues. These modifications may contribute to better extraction of the intact DNA from different bacteria present in human tissues.
Insights
A new method effectively extracts bacterial DNA from archived gastric biopsies. This technique successfully amplified the Helicobacter pylori 16S rRNA gene from all samples, enabling better genetic analysis of historical infections.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Formalin-fixed paraffin-embedded tissues are valuable for historical disease assessment.
- Established protocols for bacterial DNA extraction from archived tissues are lacking.
- Helicobacter pylori (H. pylori) infections are a significant public health concern.
Purpose of the Study:
- To develop and validate a protocol for extracting bacterial DNA from archived H. pylori-positive gastric biopsies.
- To assess the quality and quantity of extracted DNA for genetic analysis.
Main Methods:
- Modified phenol-chloroform DNA extraction protocol applied to 50 archived H. pylori-positive gastric biopsies.
- Inclusion of simultaneous proteinase K treatment and mechanical disruption for tissue digestion.
- Assessment of DNA quality via electrophoresis and PCR amplification of the H. pylori-specific 16S rRNA gene.
Main Results:
- Intact bacterial DNA was recovered from all 50 biopsy samples.
- Successful amplification of the H. pylori-specific 16S rRNA gene (519bp) was achieved from 100% of samples.
- The modified method effectively removed contaminants and improved DNA quality.
Conclusions:
- The modified DNA extraction protocol demonstrates high efficacy for recovering bacterial DNA from archived gastric tissues.
- This method facilitates reliable genetic analysis of H. pylori from historical specimens.
- The protocol may be adaptable for extracting DNA from other bacteria in human tissues.
