For any plant pathologists or fungus enthusiasts doing DNA barcoding, here’s a one-tube rapid DNA extraction method from diseased tissue or tiny immersed fruitbodies that might be useful for DNA-based identification of plant pathogens.👇
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The method was developed by Dong et al. (2022) for use in diagnosing rice blast lesions in rice (causal agent Pyricularia oryzae =Magnaporthe oryzae) using PCR and specific primers.
It involves heating a 2x2 mm fragment of a disease lesion at 95 °C for 10 minutes in an extraction buffer composed of 25 mM Tris-HCl (pH 8.7), 2.5 mM EDTA (pH 8.0), 250 mM KCl, and 0.02% Tween 20 (a detergent). 1 μL can then be used directly for PCR.
The extraction method was capable of producing PCR-ready DNA that produced amplicons of 570 bp to 1,139 bp in length without additional dilution or PCR additives. DNA extracts frozen at -20 °C were stable enough to produce PCR amplicons for three months after extraction.
Breaking down the buffer composition, it’s a pH-buffered (via Tris-HCl) high osmotic pressure (via KCl) solution containing EDTA to inhibit nucleases and a non-ionic detergent (Tween-20) to aid in wetting and cell lysis.
It's very similar to several other published methods but I don't think I've seen this exact formulation combined with heat lysis and direct PCR before. Let us know if you have!
The formulation is safe, inexpensive, and should have a good shelf-life at room temperature. It also seems very effective for rice blast lesions, suggesting that it might work well for detection of similar plant disease-causing microorganisms.
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