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

First, Trichophyton species were grown on Sabouraud dextrose agar (SDA, Difco, USA) for 10 days at 24°C in dark conditions. A sterile blade was used to scrape off the hyphae from the surface of the plate, which were transferred to a 2-mL Eppendorf tube containing 1 mL of lysis buffer (200 mM Tris-HCl, pH 8.0, with 25 mM EDTA, 0.5% [wt/vol] sodium dodecyl sulfate, and 250 mM NaCl). Cells were mechanically disrupted with a conical grinder for approximately 1 min, and then incubated at 100°C for 15 min. Next, 150 μL of 3.0 M sodium acetate buffer was added, the mixture was vortexed and incubated for 10 min at -20°C, and the solution was mixed and centrifuged for 5 min at 10,000 ×g. The supernatant was transferred to a new tube and phenol/chloroform (1:1, v/v) was used for extraction. DNA was allowed to precipitate with an equal volume of isopropanol for 10 min at -20°C and then centrifuged for 5 min at 10,000 rpm. The pellets were washed with cold 70% ethanol, dried at room temperature, resuspended in 97.5 mL of TE-buffer with 2.5 mL of RNAse (20 U/mL), and incubated for 5 min at 37°C. DNA extracts were stored at -20°C prior to use.