Specificity of a newly developed real-time PCR method for the tandem-repeat detection of azole-resistant Aspergillus fumigatus

T Toyotome1,2, D Hagiwara2,3, A Watanabe2, K Kamei2

Author address: 

1Department of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Japan 2Medical Mycology Research Center, Chiba University, Chiba, Japan 3Faculty of Life and Environmental Sciences, University of Tsukuba, Japan

Abstract: 

Purpose: Azole-resistant Aspergillus fumigatus from the environment is a growing problem in the world. Most of these strains contain characteristic tandem-repeat (TR) in cyp51A promoter. Recently, we developed a real-time PCR method to detect TR. Here in this study, we examined the specificity of the real-time PCR method to know if this system works exclusively in A. fumigatus.

Methods: Genomic DNA prepared from following clinical isolates were used in this study: 8 strains of A. niger, 4 strains of A. flavus, 3 strains of A. nidulans, and 3 strains of A. terreus. Additionally, we used genomic DNA from 14 strains of A. fumigatus including TR34/L98H strain OKH50 and TR46/Y121F/T289F strain IFM63432. For PCR, we used two cycling probes (Positive Control- or PC-probe, and TR-probe), two primers for amplification, and Cycleave PCR Reaction Mix (Takara Bio Inc.). Detection of fluorescence and analysis was performed on LightCycler 480 Instrument II System (Roche Diagnostics). ΔCt values were calculated as Ct(PC-probe) – Ct(TR-probe).

Results: ΔCt values of wild-type A. fumigatus strains ranged from −0.41 to −0.1. ΔCt value of TR azole-resistant strain OKH50 was around 0.7. The PCR reaction using DNA from non-fumigatus strains did not proceed appropriately. Calculated ΔCt values of non-fumigatus strains ranged from −4.32 to −0.78.

Conclusion: These data indicate that the newly developed real-time PCR method is highly specific for A. fumigatus, suggesting its potential to detect azole-resistant A. fumigatus among various Aspergillus spp.

2018

Full conference title: 

The 8th Advances Against Aspergillus, Lisbon Conference Center, Lisbon, Portugal
    • AAA 8th (2018)