Computational protocol: Isolation and Identification of Mushroom Pathogens from Agrocybe aegerita

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Protocol publication

[…] For DNA extraction, mycelia cultures were raised individually on PDA at 25℃ for 7 days. These pure mycelia were used for sequence analysis of the rDNA ITS region (ITS-1 region, 5.8S gene, and ITS-2 region). The genomic DNA was extracted using a DNeasy plant mini kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. Amplification and sequencing of the isolates were performed using a pair of universal primers: ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') [] for the region containing ITS1, ITS2, and the 5.8S rDNA. These primers were also used as a positive control in the subsequent diagnostic PCR. The amplification was conducted in a 20 µL reaction mixture containing 50 nM genomic DNA, 10 mM Tris-HCl, 1.5 mM MgCl2, 50 mM KCl, 0.01% gelatin, 0.2 mM dNTP, 200 ng of each primer, and 1 unit Taq DNA polymerase (Promega, Madison, WI, USA). The reaction mixtures were denatured at 94℃ for 10 min and subjected to 35 cycles of 1 min at 94℃, annealing at 55℃ for 1 min, extension at 72℃ for 1 min, and a final extension step of 7 min at 72℃. The amplified PCR product was separated on a 1.5% agarose gel, followed by purification with a DNA purification kit (Core-one™; Core-Bio, Seoul, Korea), according to the manufacturer's instructions. Both amplicon strands were sequenced using the same primers, reactions were monitored with BigDye Terminator Cycle Sequencing kits (Applied Biosystems, Foster City, CA, USA), and run on an ABIPRISM 3130 automated DNA sequencer (Applied Biosystems), as described previously []. The rDNA ITS sequence data were analyzed using the DNASTAR program (DNASTAR Inc, Madison, WI, USA) and aligned by the CLUSTAL W method []. MEGA ver. 4.0 was used for the phylogenetic analysis [, ]. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Pleurotus ostreatus, Trichoderma atroviride