70F, CBS 700.71 and CBS 700.68 were used as negative controls in tests with non-orthologous taxa. The concordance of RCA results and identification by multilocus

sequencing was 100%. Products of the RCA reaction were visualised by electrophoresis on 1% agarose gels. With exonucleolysis, positive responses showed ladder-like patterns after RCA, whereas with negative results the background remained clean. When exonucleolysis was omitted, a single, weak or strong band was visible on negative lanes, representing a non-specific band that did not interfere with the RCA reaction. Sensitivity testing showed that RCA yields positive results in wide ranges of amplicon concentrations down to 3.2 × 105 copies of amplicon (Fig. 2). rDNA ITS is a sufficient barcoding marker in Mucorales, because interspecific distances tend to be relatively large compared to e.g. more recently evolved BAY 80-6946 mouse ascomycetes.[11] In addition, the majority of clinically relevant taxa are located in distantly related clades. The main exception is with R. arrhizus var. arrhizus and R. arrhizus var. delemar which differ in 3 bp in ITS, show occasional interbreeding and have been considered to be varieties of a single species rather than separate species.[21] RCA

reportedly has a specific detection limit of single nucleotide [17] and thus should be able to differentiate between these groups. Our results showed that this was indeed the case (Fig. 1). The purpose of the present BAY 73-4506 study was to establish a screening method based on the RCA enabling rapid detection, with specificity down to few nucleotide differences and assess the limits Fluorouracil mouse of this molecular method. We found specificity of 100%, and high sensitivity. RCA is a robust

and simple isothermal DNA amplification technique allowing rapid detection of specific nucleic-acid sequences with no need of sequencing and can be performed within 2 h, and is therefore applicable for rapid and economic screening purposes.[16, 17] Specially designed padlock probes hybridise to a target DNA or RNA and permit the detection of single nucleotide mismatch and prevent non-specific amplification, a common risk factor in conventional PCR. To date, RCA has been used for different fungi, such as Cryptococcus, Trichophyton, Candida, Aspergillus, Talaromyces marneffei, Scedosporium and black yeast.[17] In Mucorales no cross reactivity was observed within tested strains. RCA is particularly suited for high throughput applications. Wide ranges of amplicon concentrations yield positive results. The amplification product can be visualised by agarose gel electrophoresis, but also in gel-free systems using fluorescence staining of amplified product by SYBR Green in combination with UV-transillumination, and this can add to the speed and ease of the test. RCA is practical for detection of low copy number DNA. The method can be performed with a variety of DNA polymerases compared to direct PCR.