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Optimizing a modified cetyltrimethylammonium bromide protocol for fungal DNA extraction: Insights from multilocus gene amplification

Summary

This research improves how scientists extract DNA from fungi found in dried fruits and other sources. The modified CTAB method is faster, cheaper, and produces higher quality DNA than commercial kits, without requiring special equipment like liquid nitrogen. The extracted DNA works well for identifying fungal species and detecting fungal diseases in fish, plants, and humans, making it valuable for both research and medical diagnostics.

Background

Genomic DNA extraction is critical for fungal molecular studies, but efficient methods that minimize DNA degradation and contamination remain challenging due to fungal cell wall composition. Various extraction methods exist with different yields and quality outcomes. The CTAB method has been adapted for fungal applications to overcome limitations of commercial kits.

Objective

To evaluate and optimize a modified CTAB protocol for fungal DNA extraction from various species including Aspergillus, Penicillium, Alternaria, Dothiorella, and Fusarium. The study aimed to assess DNA yield, quality, extraction efficiency, and successful multilocus gene amplification compared to commercial extraction kits.

Results

The modified CTAB method yielded significantly higher DNA concentrations (mean 267.67 ± 20.69 ng/µL) compared to HiMedia and Qiagen kits (p < 0.001). All three cell crushing methods produced similar results without significant differences. DNA fragment sizes ranged from 30-60 kb, and successful amplification was achieved for all five gene loci in most isolates, with sequence similarity of 95-100% for various fungal species.

Conclusion

The modified CTAB protocol proves cost-effective and rapid for fungal DNA extraction without requiring liquid nitrogen or bead beating. The protocol successfully produces high-quality, high-molecular-weight DNA suitable for downstream applications including PCR amplification and MLST analysis, with potential applications in fungal disease diagnosis for fish, plants, and humans.
  • Published in:Open Life Sciences,
  • Study Type:Experimental Method Optimization,
  • Source: PMID: 39926474
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