Evaluation of two molecular techniques for rapid detection of the main dermatophytic agents of XXXX .

@entity14476 @entity3100
BACKGROUND: @entity3100 is very common in Western China, with the most widespread aetiological agent being Trichophyton violaceum, while @entity14476 is prevalent in the remainder of China. Conventional diagnostics and internal transcribed spacer (ITS) sequencing analyses have proven relatively limited due to the close phylogenetic relationship of anthropophilic dermatophytes. Therefore, alternative molecular tools with sufficient specificity, reproducibility and sensitivity are necessary. OBJECTIVES: To evaluate two molecular techniques [multiplex ligation-dependent probe amplification (MLPA) and rolling circle amplification (RCA)] for rapid detection of the aetiological agents of @entity3100 , T. violaceum and @entity14476 . METHODS: Probes of RCA and MLPA were designed with target sequences in the rDNA ITS gene region. Strains tested consist of 31 T. violaceum, 22 @entity14476 and 24 reference strains of species that are taxonomically close to the target species. RESULTS: The specificity and reproducibility of RCA and MLPA in detection of T. violaceum and @entity14476 were both 100% in both species. Sensitivity testing showed that RCA was positive at concentrations down to 1  68 * 10(6) copies of DNA in the TvioRCA probe, and 2  7 * 10(8) copies of DNA in McRCA. MLPA yielded positive results at concentrations of DNA down to 1  68 * 10(1) copies in the TvioMLPA probe and 2  7 * 10(2) in McMLPA. CONCLUSIONS: The two techniques were sufficiently specific and sensitive for discriminating the target DNA of T. violaceum and @entity14476 from that of closely related dermatophytes. RCA and MLPA are advantageous in their reliability and ease of operation compared with standard polymerase chain reaction and conventional methods.
