Use of Padlock Probes and Rolling Circle Amplification (RCA) for Rapid Identification of Trichophyton Species, Related to Human and Animal Disorder

Zakeri, Hamideh; Shokohi, Tahereh; Badali, Hamid; Mayahi, Saba; Didehdar, Mojtaba

The ribosomal DNA internal transcribed spacers (i.e., the ITS region of rDNA) were amplified using the universal primers ITS1 (5'-TCC-GTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') (13) . PCR reactions were performed on a TC-312 thermal cycler (Techne, Duxford, Cambridge, United Kingdom) in 25-mL volumes containing 25 ng of template DNA, 2.5 mL of reaction buffer (0.1 M Tris-HCl, pH 8.0, 0.5 M KCl, 15 mM MgCl2, 0.1% gelatin, and 1% Triton X-100), 0.2 mM of each dNTP, and 2.0 U of Taq DNA polymerase. Amplification was performed as follows: 2 min at 94°C for primary denaturation, followed by 35 cycles at 94°C (45 s), 52°C (30 s), and 72°C (120 s), with a final 7-min extension step at 72°C. PCR products were visualized by 1.5% (w/v) agarose gel electrophoresis in TBE buffer, stained with ethidium bromide (0.5 μg/mL), and photographed under UV transillumination.