Development and validation of LAMP primer sets for rapid identification of Aspergillus fumigatus carrying the cyp51A TR46 azole resistance gene

Abstract

Infections due to triazole-resistant Aspergillus fumigatus are increasingly reported worldwide and are associated with treatment failure and mortality. The principal class of azole-resistant isolates is characterized by tandem repeats of 34 bp or 46 bp within the promoter region of the cyp51A gene. Loop-mediated isothermal amplification (LAMP) is a widely used nucleic acid amplification system that is fast and specific. Here we describe a LAMP assay method to detect the 46 bp tandem repeat insertion in the cyp51A gene promoter region based on novel LAMP primer sets. It also differentiated strains with TR₄₆ tandem repeats from those with TR₃₄ tandem repeats. These results showed this TR₄₆-LAMP method is specific, rapid, and provides crucial insights to develop novel antifungal therapeutic strategies against severe fungal infections due to A. fumigatus with TR₄₆ tandem repeats.

Figure 1

Genetic information for the design of the LAMP primer sets. (A) Schematic illustration of cyp51A gene showing LAMP primer positions and corresponding sequences of TR46 bp promoter tandem repeat compared to wild-type sequences. (B) Primers F3, F2, F1, B1, B2, and B3 show primer sequence positions. Sequences of some primers are complementary, as shown in Table 2. See LAMP primer and methods, which are shown in Refs.^,.

Figure 2

Illustration of tandem repeat regions of cyp51A genes used in this experiment. (A) Tandem repeat unit of promoter genes of TR₃₄ and TR₄₆. (B) Tandem repeat: 34 bp (double) and 46 bp (double or triple), and cyp51A gene associated point mutation place.

Figure 3

Schematic figure of TR₄₆ LAMP primer amplification site in comparison with those of wild type and TR₃₄. The nucleotide sequence is targeted for the promoter region for the TR₄₆ resistance gene (between TR₄₆-1 and TR₄₆-2). The sequence in this part corresponds to the B2 sequence in Table 2.

Figure 4

Comparative amplification profiles of A. fumigatus wild type and environmental or clinical azole-resistant isolates with or without TR46 double or triple 46 bp promoter repeats in cyp51A gene by a newly developed LAMP primer sets. The dotted curve shows the amplification by control strain (IFM 63432). (A-i) DNA amplification profiles using 30 strains of A. fumigatus wild type. DNA amplification was not confirmed in all wild-type strains tested (30 strains). Among 30 wild-type strains, IFM 62918 strain was used as a negative control strain (no amplification). (A-ii) DNA amplification was confirmed by five TR₄₆² strains (IFM63432, BE1-2, BE1-4, BE3-5, BE3-6), which have double 46 bp promoter repeats. (A-iii) DNA amplification was confirmed by three TR₄₆³ strains (BE1-1, W1-4, W2-12-1), which have triple 46 bp promoter repeats. (B-i) DNA amplification was not confirmed by two TR₃₄² strains (IFM64460, IFM64733), which have duplicate 34 bp promoter repeats with one mutation in the one coding region (L98H). The dotted line shows amplification by the control strain. (B-ii) DNA amplification was not confirmed by one TR₃₄² strain (3-1-B), which has duplicate 34 bp promoter repeats with multi-mutations in the four coding regions (L98H/T289A/I364V/G448S). The dotted line shows amplification by the control strain.

Figure 5

Experimental results of the detection limit of the TR₄₆ LAMP assay. The detection limit reaction was carried out using 10⁷ to 10 copies of plasmid DNA per reaction. The detection limit was measured within 60 min.