A novel broad allele-specific Taqman real-time PCR method to detect triazole-resistant strains of Aspergillus fumigatus, even when in very low percentage of the triazole-resistant and -susceptible strain mixture.

A novel broad allele-specific Taqman real-time PCR method to detect triazole-resistant strains of Aspergillus fumigatus, even when in very low percentage of the triazole-resistant and -susceptible strain mixture.

J Clin Microbiol. 2019 Jul 17;:

Authors: Wang Q, Kontoyiannis DP, Li R, Chen W, Bu D, Liu W

Abstract
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 Therefore, it is crucial to develop robust methods to identify triazole-resistant strains of A. fumigatus, even from the mixture 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, TR46, G54W, 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 (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 resistance in the mixture of triazole-resistant and -susceptible strains of A. fumigatus The method had high efficiency and sensitivity (300 fg/well, corresponding to about 100 CFUs per reaction). It could promptly detect triazole resistance in a panel of 30 clinical strains of A. fumigatus within about 6 hours. 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 robust to detect cyp51A-associated resistance alleles even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus and should have important clinical applications.

PMID: 31315952 [PubMed – as supplied by publisher]

Source: Industry