Polymerase chain reaction

The extracted crude DNA was used as a template for polymerase chain reaction (PCR) without dilution or further purification. The universal fungal primer set (Invitrogen, Inc., Shanghai, China) consisting of ITS1F (5′-CTT GGT CAT TTA GAG GAA GTA A-3′)28 and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′)29 was used for amplification of fungal internal transcribed spacer (ITS) regions. The 5′ end of ITS1F was labeled with the fluorescent dye 6-FAM. PCR reaction mixtures (50 μL) contained 2 μL of each primer (10 μmol/L), 1 μL of bovine serum albumin (0.4 μg/μL), 25 μL of 2× Taq MasterMix (Cowin Biotech, China), 2 μL of template DNA, and 18 μL of sterilized water. The cycling parameters were 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 7 min.