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.

Invasive aspergillosis caused by triazole-resistant strains of is a growing public health concern, as is the occurrence of mixed infections with triazole-resistant and -susceptible strains. Therefore, it is crucial to develop robust methods to identify triazole-resistant strains of , even in mixtures of triazole-resistant and -susceptible strains of 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 TR (a 34-bp tandem repeat in the promoter region), TR, G54W (a change of G to W at position 54), G54R, L98H, Y121F, and M220I mutations in the gene of 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 alleles. We used this method to detect triazole-resistant clinical strains of with a variety of gene mutations, as well as the triazole-resistant strains in mixtures of triazole-resistant and -susceptible strains of 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 within about 6 h. It could also detect -associated resistance alleles, even in mixtures containing only 1% triazole-resistant strains. These results suggest that this method is robustly able to detect -associated resistance alleles even in mixtures of triazole-resistant and -susceptible strains of and that it should have important clinical applications.