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

Recently, isothermal techniques, such as loop-mediated amplification and RCA, have been applied for rapid identification. The use of circularizable oligonucleotides or padlock probes is based on the rolling replication of short single-stranded DNA circles by certain DNA polymerases under isothermal conditions to detect target nucleic acid sequences, including SNPs, with high specificity (9) (10) (11) (12) (13) (14) (15) (16) . Such probes comprise two sequences that are complementary to the 5' and 3' termini of the target sequence joined by a genetic linker region. Upon hybridization to the target, the two probe ends become juxtaposed and are joined by DNA ligase to form a closed molecule. The intensity of the signal generated by the probe is then increased exponentially by hyper branching or RCA, and 109-fold signal amplification of each circle can be achieved within 90 min (8, 13, 17) . The RCA technique was initially established by Fire and Xu (18) and Liu et al. (19) and is an isothermal in vitro DNA amplification method. It is one of a series of robust and simple techniques for distinguishing closely related taxa at the species as well as the strain level. RCA is a rapid (requiring less than 1 working day), specific (to the single-nucleotide level), and economical (requiring minimal equipment) tool for fungal screening (20, 21) .