2.2.2. Acid Nucleic Detection through Quantitative Real-Time PCR (qPCR)

Three different target genes were amplified with qPCR approach to investigate the differences in acid nucleic detection: a long-repeated fragment (Large Sub-Units, LSU gene of 939 bp), a short-repeated fragment of the same gene (LSU gene of 330 bp) and a small but non-repeated fragment in the genome (β-actin gene of 330 bp). qPCR was performed with a BioRad CFX96 real time PCR detection system (BioRad, Hercules, CA, USA) using primers targeting the fungal LSU rRNA gene and the β-actin gene: LR0R (ACCCGCTGAACTTAAGC, [30]) and LR5 (TCCTGAGGGAAACTTC, [31]), and ACT512- F (ATGTGCAAGGCCGGTTTCGC3) and ACT783-R (TACGAGTCCTTCTGGCCCAT) [32], respectively, each at 5 pmol final concentration.

The primers LR0R-LR5 and LR0R-LR3 were used to amplify a 939 bp and 300 bp products, respectively, spanning the LSU region of rRNA encoding genes. The standard qPCR cycling protocol for both products, consisting of a denaturation step at 94 ◦C for 5 min, followed by 35 cycles of denaturing at 94 ◦C for 45 s, annealing at 52 ◦C for 30 s, and elongation at 72 ◦C for 2 min, was performed. The primers ACT512-F and ACT783-R are used to amplify a 330 bp product spanning the β-actin gene. The standard qPCR cycling protocol, consisting of a denaturation step at 95 ◦C for 10 min, followed by 35 cycles of denaturation at 95 ◦C for 15 s, annealing at 61 ◦C for 20 s, and elongation at 72 ◦C for 15 s, was performed. Fluorescence measurements were recorded at the end of each annealing step. After 35 cycles, a melt curve analysis was performed by recording changes in fluorescence as a function of raising the temperature from 60–90 ◦C in 0.5 ◦C per increments. All tests were performed in triplicate.
