A Simple Method To Detect Point Mutations in Aspergillus fumigatus cyp51A Gene Using a Surveyor Nuclease Assay

Abstract

One of the main mechanisms of azole resistance of Aspergillus fumigatus is thought to be a reduction in the drug's affinity for the target molecule, Cyp51A, due to its amino acid mutation(s). It is known that the azole resistance pattern is closely related to the mutation site(s) of the molecule. In this study, we tried to develop a simple and rapid detection method for cyp51A mutations using the endonuclease Surveyor nuclease. The Surveyor nuclease assay was verified using several azole-resistant strains of A. fumigatus that possess point mutations in Cyp51A. For validation of the Surveyor nuclease assay, blind tests were conducted using 48 strains of A. fumigatus (17 azole-resistant and 31 azole-susceptible strains). The Surveyor nuclease assay could rapidly detect cyp51A mutations with one primer set. Also, all the tested strains harboring different cyp51A single point mutations could be clearly distinguished from the wild type. The Surveyor nuclease assay is a simple method that can detect cyp51A mutations rapidly.

Keywords: Aspergillus fumigatus; azole resistance; cyp51A.

FIG 1

Efficiency of cleavage by SN. The 1,661-bp amplicons were PCR amplified. The G54W mutant amplicon was annealed in combination with the reference amplicon (wild-type cyp51A). SN treatment was performed at 42°C for 20, 30, 40, 50, and 60 min. The cleavage products were 1,480 and 181 bp. The control was annealed alone with the reference amplicon.

FIG 2

Detection of major mutations by SN. Six major mutations were tested by SN assay. (a) Detection patterns of the G54 mutation. Lane 1, control; lane 2, G54W (cleavage fragments, 181 and 1,480 bp); lane 3, G54R (181 and 1,480 bp); lane 4 G54V (182 and 1,479 bp). (b) Detection patterns of the M220 mutation. Lane 5, control; lane 6, M220K (fragments, 751 and 910 bp); lane 7, M220I (752 and 909 bp); lane 8, M220V (750 and 911 bp). (c) Detection patterns of Y120F/T289A (lane 10; the fragments were 454, 503, and 704 bp), G448S (lane 11; 227 and 1,434 bp), and L98H (lane 12; 385 and 1,276 bp) mutations. In the Y120F/T289A lane, incomplete cleavage fragments (957 and 1,207 bp) can be observed. Lane 9, control. (d) Lane 14, 5SNP (Af293) detection pattern (many cleavage fragments were detected); lane 13, control.