Metagenome Extraction of Respiratory Samples and Evaluation of Bacterial Diversity via PCR-DGGE

Introduction and objective: DNA extraction is a fundamental procedure in any culture independent approach to determine microbial diversity. Methods used for this purpose should accurately reflect the diversity. Due to low microorganism amount in some clinical samples including respiratory samples, DNA extraction method is problematic to get an appropriate yield. In this study, three methods of DNA extraction from pharyngeal swab were evaluated. Then, PCR and PCR-DGGE techniques were carried out for these DNA extractions. Materials and methods: Pharyngeal swabs were transferred to laboratory in TSB tubes. In the first method, DNA was extracted by regular boiling technique. In the second method, chloroform technique without phenol was used. In the third method, lysis buffer was at first added to pharyngeal samples, and further steps followed for second method. To increase the DNA yield, a physical cell lysis step using glass beads was added to all methods. Afterwards, two sets of universal bacterial primers for 16s rDNA (27f , 1492r and GC-341f , 782r) were used and the final product of the second pair primers surveyed with PCR-DGGE technique. Results: Highest DNA concentration was obtained from the first method, and lowest DNA concentration was achieved in the third method. However, PCR products were successfully achieved using DNA extraction of the third method when the number of bacterial cells was low. PCR-DGGE result of each sample shows a similar pattern for all methods. Conclusion: Similar bacterial diversity was reflected by different extraction methods. Although using the first method which is fast, economical and simple, is recommended for samples with high number of bacteria, for samples with low number of bacteria third method is recommended.

کلیدواژه ها:

DNA extraction ، Bacterial diversity ، PCR-DGGE