Iranian journal of pathology · 2023
An Efficient and Easy- to- Use Method for Extraction of H. pylori DNA from Archival Formalin-Fixed and Paraffin-Embedded Gastric Tissues.
СтатьяНазвание и аннотация: Перевод в очереди
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Saniee Parastoo, Ghadersoltani Paria, Noroozpour Masoumeh, Sadjadi Alireza
Аннотация
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.
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.