Rapid, high-throughput, multiplex, real-time PCR for identification of mutations in the cyp51A gene of Aspergillus fumigatus that confer resistance to itraconazole

Abstract

Aspergillus fumigatus is an important cause of life-threatening invasive fungal disease in patients with compromised immune systems. Resistance to itraconazole in A. fumigatus is closely linked to amino acid substitutions in Cyp51A that replace Gly54. In an effort to develop a new class of molecular diagnostic assay that can rapidly assess drug resistance, a multiplexed assay was established. This assay uses molecular beacons corresponding to the wild-type cyp51A gene and seven mutant alleles encoding either Arg54, Lys54, Val54, Trp54, or Glu54. Molecular beacon structure design and real-time PCR conditions were optimized to increase the assay specificity. The multiplex assay was applied to the analysis of chromosomal DNA samples from a collection of 48 A. fumigatus clinical and laboratory-derived isolates, most with reduced susceptibility to itraconazole. The cyp51A allelic identities for codon 54 were established for all of the strains tested, and mutations altering Gly54 in 23 strains were revealed. These mutations included G(54)W (n = 1), G(54)E (n = 12), G(54)K (n = 3), G(54)R (n = 3), and G(54)V (n = 4). Molecular beacon assay results were confirmed by DNA sequencing. Multiplex real-time PCR with molecular beacons is a powerful technique for allele differentiation and analysis of resistance mutations that is dynamic and suitable for rapid high-throughput assessment of drug resistance.

FIG. 1.

cyp51A allele discrimination by molecular beacons. Real-time PCR was used to evaluate the specificity of eight different molecular beacons designed to distinguish the wild-type allele and seven mutant alleles. Each panel shows an individual molecular beacon and its relevant Gly54 allele recognition sequence. Real-time PCRs were performed with separate 500-bp templates corresponding to the GGG (*), GAA (▾), AAG (□), GAG (⋄), AGG (▪), GTG (♦), and TGG (▴) cyp51A alleles and a blank (no DNA; •) with the indicated specific molecular beacons. Each panel shows a composite representation of eight separate template reactions with the same molecular beacon.

FIG. 2.

Multiplex assay formats. (A) Graphic output of the Stratagene Mx4000 software for the multiplex single-tube assay. The top semicircle is highlighted when the FAM signal is observed, reporting the presence of the wild-type cyp51A allele. The bottom semicircle is highlighted when the HEX signal is observed, reporting the presence of any of seven mutant cyp51A alleles. (B) Graphic output from the Stratagene Mx4000 software for the multiplex double-tube assay. Each quadrant of the eight total sectors in the two circles represents an allele-specific molecular beacon labeled with HEX, ROX, Q670, or FAM, as indicated. The top right sector of the upper circle is highlighted when the FAM signal is observed, reporting the presence of the wild-type cyp51A allele. All other sectors are highlighted when the HEX, ROX, Q670, or FAM signal is observed, reporting the presence of a specific mutant cyp51A allele.