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

Summary

Researchers developed an improved method for extracting fungal DNA that is faster, cheaper, and safer than current commercial kits. The new protocol works by using a special chemical solution and simple crushing tools instead of expensive equipment like liquid nitrogen or bead beaters. They tested it on 40 different fungal species found in dry fruits and successfully identified them all using genetic markers, showing the method could help diagnose fungal diseases in plants, animals, and humans.

Background

Genomic DNA extraction is a crucial step in fungal molecular studies, and various extraction methods differ in yield and quality. The modified CTAB method has emerged as effective for extracting high molecular weight DNA with minimal protein contamination from fungal species.

Objective

To evaluate the efficiency, cost-effectiveness, and efficacy of a modified CTAB protocol for fungal DNA extraction and compare it with commercial kits, while assessing successful amplification of multiple gene loci for fungal identification.

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). DNA fragment sizes ranged from 30-60 kb. All three cell crushing methods showed similar DNA yields with no significant differences. Successful PCR amplification was achieved across multiple gene loci in 40 fungal isolates, with sequence similarity of 93-100% to known species.

Conclusion

The modified CTAB protocol is cost-effective, rapid, and eliminates the need for liquid nitrogen or bead beating while maintaining high DNA quality. The protocol successfully produces high-purity genomic DNA suitable for PCR amplification and MLST analysis, with potential applications in fungal disease diagnosis including plant pathogens, human fungal infections, and fish fungal diseases.
  • Published in:Open Life Science,
  • Study Type:Experimental Method Evaluation,
  • Source: PMID: 39926474, DOI: 10.1515/biol-2022-1006
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