A Novel Broad Allele-Specific TaqMan Real-Time PCR Method To Detect Triazole-Resistant Strains of Aspergillus fumigatus, Even with a Very Low Percentage of Triazole-Resistant Cells Mixed with Triazole-Susceptible Cells

J Clin Microbiol. 2019 Aug 26;57(9):e00604-19. doi: 10.1128/JCM.00604-19. Print 2019 Sep.


Invasive aspergillosis caused by triazole-resistant strains of Aspergillus fumigatus is a growing public health concern, as is the occurrence of mixed infections with triazole-resistant and -susceptible A. fumigatus strains. Therefore, it is crucial to develop robust methods to identify triazole-resistant strains of A. fumigatus, even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus In this work, we developed a robust, highly selective, and broad-range allele-specific TaqMan real-time PCR platform consisting of 7 simultaneous assays that detect TR34 (a 34-bp tandem repeat in the promoter region), TR46, G54W (a change of G to W at position 54), G54R, L98H, Y121F, and M220I mutations in the cyp51A gene of A. fumigatus The method is based on the widely used TaqMan real-time PCR technology and combines allele-specific PCR with a blocking reagent (minor groove binder [MGB] oligonucleotide blocker) to suppress amplification of the wild-type cyp51A alleles. We used this method to detect triazole-resistant clinical strains of A. fumigatus with a variety of cyp51A gene mutations, as well as the triazole-resistant strains in mixtures of triazole-resistant and -susceptible strains of A. fumigatus The method had high efficiency and sensitivity (300 fg/well, corresponding to about 100 CFU per reaction mixture volume). It could promptly detect triazole resistance in a panel of 30 clinical strains of A. fumigatus within about 6 h. It could also detect cyp51A-associated resistance alleles, even in mixtures containing only 1% triazole-resistant A. fumigatus strains. These results suggest that this method is robustly able to detect cyp51A-associated resistance alleles even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus and that it should have important clinical applications.

Keywords: A. fumigatus; allele-specific real-time PCR; cyp51A mutations; mixed infection; triazole resistance.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Antifungal Agents / pharmacology*
  • Aspergillosis / diagnosis*
  • Aspergillus fumigatus / drug effects
  • Aspergillus fumigatus / genetics
  • Aspergillus fumigatus / isolation & purification*
  • Cytochrome P-450 Enzyme System / genetics
  • Drug Resistance, Fungal*
  • Fungal Proteins / genetics
  • Genotyping Techniques / methods
  • Humans
  • Molecular Diagnostic Techniques / methods*
  • Real-Time Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Time Factors
  • Triazoles / pharmacology*


  • Antifungal Agents
  • Fungal Proteins
  • Triazoles
  • Cytochrome P-450 Enzyme System
  • cytochrome P-450 CYP51A, Aspergillus