C-217. The Use of High-Resolution Melt on the Rotor-GeneTM 6000 to Characterize Codon 54 of the cyp51A Gene of Aspergillus fumigatus

M. J. Tuohy1, V. Reja2, S. Park3, D. S. Perlin3, M. Wnek1, B. Yen-Lieberman1;
1Cleveland Clin., Cleveland, OH, 2Corbett Research, Sydney, AUSTRALIA, 3UMDNJ-New Jersey Med. Sch., Newark, NJ.

Background: High-Resolution Melt (HRM) is a new method used to characterize real-time amplification products. It requires specialized dyes and instrumentation, but not probes, and it has been used successfully for single nucleotide polymorphism (SNP) genotyping. We evaluated this technology for characterizing the codon for glycine 54 in the cyp51A gene for isolates of Aspergillus fumigatus. Mutations in this gene result in reduced susceptibility to itraconazole and posaconazole. Methods: Thirteen well-characterized strains representing wild type Gly54 (GGG) and 6 mutant alleles of cyp51A including Arg54 (AGG), Lys54 (AAG), Val54 (GTG), Trp54 (TGG) or Glu54 ( GAG and GAA) were tested. In addition, 12 clinical strains of A. fumigatus were tested as unknowns. DNA was obtained using glass bead lysis, heating at 100°C, and then extracting with a MagNA Pure LC (Roche Diagnostics, Indianapolis, IN. ) Primers were designed to amplify a 107 bp fragment of the cyp51A gene covering codon 54. Real-time PCR and HRM were performed using SensiMix HRM™ (Quantace, London, England) and the Rotor-Gene™ 6000 instrument (Corbett Research, Sydney, Australia). Rotor-Gene software was used for the data analysis. All genotypes were confirmed by pyrosequencing (Biotage, Uppsala, Sweden), as previously described. Results: The wild type 54th codon GGG of cyp51a was clearly differentiated from all the mutant strains of GAG, GAA, AAG, AGG, GTG, TGG using HRM. However, melting temperatures were not specific enough to accurately characterize each mutant allele beyond non-wild type. The 12 clinical strains tested as wild type GGG, and were verified by pyrosequencing. Conclusions: Our HRM assay using the Rotor-Gene 6000 was very easy to perform and critical genotyping information was obtained quickly. Using this new technology, genetic mutations in codon 54 of the cyp51a gene of A. fumigatus can be identified as non-wild type, conferring probable reduced susceptibility to itraconazole and posaconazole.