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

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.

www.nature.com/scientificreports/ of patients with invasive aspergillosis caused by azole-resistant strains has been reported 12,13 . Thus, a rapid and specific method to identify the presence of TR would contribute to faster therapeutic decision-making 14 .
As one of the promising diagnostic tools for the azole-resistant A. fumigatus, loop-mediated isothermal amplification (LAMP) for the development of improved DNA-based diagnostic kits has been reported 15 . In general, the LAMP method was found to be similar or superior to the standard PCR method, more specific, lower-cost, and easier to perform. LAMP-based approaches have been applied to a wide range of samples, such as whole blood, paraffin-embedded tissues, and various microbial pathogens 16,17 . In this paper, we report a novel LAMP assay method that selectively detects triazole resistant A. fumigatus strains due to the presence of double TR46(TR 46 2 ) or triple TR 46 (TR 46 3 )in the cyp51A promoter region.

Results
Antifungal susceptibility tests. Drug susceptibilities of 41 A. fumigatus strains against azole drugs itraconazole and voriconazole are shown in Table 1. Thirty strains designated as wild type were isolated from clinical specimens, and they were confirmed to be susceptible to itraconazole and voriconazole. The remaining 11 strains (TR 34 and TR 46 ) were resistant to voriconazole, and most of them showed MIC values of > 8 μg/mL against voriconazole. Among the 11 strains, 2 strains (IFM64460 with TR34/L978H and IFM64733 with TR34/LH98H) were resistant to itraconazole, and the remaining 9 strains were susceptible to itraconazole (Table 1).
Primer design. The most crucial step in the LAMP assay is the design of primers. In the LAMP assay, six primers are necessary to amplify the targeted region under isothermal condition. First, we inspected the promoter region (− 461 bp to − 296 bp counted from start codon) of the cyp51A gene to select a set of primer sequences that specifically amplify the repeated 46 bp sequence in strains with a TR 46 mutation (Figs. 1 and 2). To enable specific amplification against repeated TR 46 sequences, B2 was set on the joint of two 46 bp sequences. Then, another five sequences for primer sets were chosen in the target region, according to the standard criteria, to obtain a specific and rapid LAMP primer set in the LAMP assay. Namely, six primers (F1, F2, F3, B1, B2, B3) that target six specific regions of a DNA template of the TR 46 gene of cyp51A were selected, and in addition, two loop primers (LF, LB) were also chosen to accelerate the reaction (Fig. 1). Several new candidates of LAMP primers were designed based on the above information and their utility tested. From those, one useful LAMP primer set based on the detection of TR 46 regions in the cyp51A gene was selected (Table 2). In this LAMP method, the primers were selected based on the criteria that amplification started within about 30-50 min, and maximum amplification was completed within 70-80 min. Nucleotide sequence of promoter region for resistance gene of LAMP primer sets to detect the resistance gene was shown in Fig. 3. This primer amplifies between consecutive 46 bp sequences between TR 46 -1 bp and TR 46 -2 bp. The base sequence in this part corresponds to the B2 sequence in Table 2.
Validation of LAMP primer sets for TR 46 . The specificity of the primer sets was tested using various types of A. fumigatus strains, such as wild isolates and environmental or clinical azole resistance isolates (Fig. 4).
In this study, IFM63432 and IFM62918 strains were used as positive and negative control strains, respectively. As shown in Fig. 4A (Table 1), namely strains IFM64460 and IFM64733 (with mutation of TR 34 /L98H) and strain 3-1-B (with mutations of TR 34 /L98H/ Y289/T289A/I364V/G448S), DNA amplification was not observed (Fig. 4B-i,B-ii). These results also suggested that the present LAMP primer could not detect TR 34 2 drug-resistant strains regardless of their point mutation site in the cyp51A gene ( Fig. 4B-i,B-ii). These studies confirmed that the newly established TR 46 LAMP primer set was specific for A. fumigatus strains with TR of double or triple 46-bp promoter tandem repeats in the cyp51A gene. The sensitivity of the TR 46 LAMP assay was verified. The detection limit was 1 × 10 4 copies per reaction in 60 min. In the 80 min reaction, 10 2 copies per reaction were also detected (Fig. 5).

Discussion
Azole antifungals mainly inhibit the ergosterol biosynthetic pathway by targeting the cytochrome P450-dependent enzyme lanosterol 14-α-demethylase, encoded by cyp51A in molds. Resistance to this class of drugs in the major human pathogen A. fumigatus is emerging and reaching levels to prevent their clinical use 6 . Advances in recent molecular genetic technologies such as real-time PCR have introduced various proper diagnostic assay methods into the fields in azole-resistant mechanism analysis. The LAMP assay described here has advantages of high sensitivity and specificity, low costs, and short amplification time. In addition, there have been no reports using LAMP techniques to study azole-resistant mechanisms in A. fumigatus by the strains with TR 46 in the cyp51A promoter region.
Recently Yu Shan-Ling et al. 18 reported a similar rapid technique to detect azole-resistant strains due to amplification of a TR of a 34 bp (TR 34 ) and a 46 bp (TR 46 ) within the promoter region of cyp51A of A. fumigatus. However, here we used a newly designed TR 46 LAMP primer set different from those reported by Yu Shan-Ling et al. 18 . Compared to experiments such as Yu Shan-Ling et al. 18 19 but not to itraconazole. Further detailed drug susceptibility mechanism study against TR 34 strain (3-1-B: TR 34 /L98H/T289A/I364V/G448S) is of interest.
The high specificity and rapidity of the LAMP assay are achieved by applying four primers that target six regions of a DNA template, and two loop primers (LF, LB) to accelerate the reaction. In this study, we succeeded in designing valuable TR 46 LAMP primer sets to detect specifically a TR 46 within the promoter regions www.nature.com/scientificreports/  Table 2. See LAMP primer and methods, which are shown in Refs. 19,20 .   20 . The LAMP primer we designed was able to detect both two copies of the TR46 tandem repeat and three copies of the TR 46 . Moreover, these amplification curves (as well as the starting point) were similar. The BIP (B1 + B2 complementary; Fig. 1) of the primer we designed is TR-specific. B1 is designed at the boundary where the repeat unit is inserted, and B2 is designed at the boundary between the repeat units. In addition, B3 is designed on a repeat unit. Based on the results of strains having double repeat and triple repeat, it was suggested that the primer used this time may be able to detect even if the number of repeats increases, such as TR 46 4 . It is widely known that exposure to azole fungicides resulted in the emergence of azole-resistant strains with tandem repeats in the promoter region of cyp51A gene 8,9 . For this reason, epidemiological studies such as the incidence of azole-resistant strains in the environment are essential. Many environmental and clinical isolates need to be screened to generate epidemiological data, such as the frequency of detection of azole-resistant A. fumigatus. The method developed in this study would be an easy-to-use screening procedure.
Since the LAMP assay developed in the present study is a one-step and rapid detection method, coupled with its high reliability and ease of use, it can prompt detect specific drug-resistant genes due to TR 46 in A. fumigatus in the clinical laboratory setting. Thus, early detection of infections due to TR 46 drug-resistant strains in A. fumigatus might be helpful to guide the early start of corrective and effective antifungal therapy. Table 2. Sequence information of newly designed TR46-LAMP primer sets in the present experiment.

F3
CAT ATG TTG CTC AGC GGC AG   B3  CAA CTT TCA TTC GGC TCA GCA   FIP (F1 complementary + F2)  GAG TTT TTT CTA GAG GAG AAC AGT G-CAC GTG CGT AGC AAG GGA   BIP (B1 + B2 complementary)  CAC CAC TTC AGA GTT GTC TAG AAT -ACC GCG TGA TTC TAG ACA ACT TTC   LF  ATT ATT CAG AGT GCT TCT TTC CTT C   LB GGT CCG GAT GTG TGCTG Figure 3. Schematic figure of TR 46 LAMP primer amplification site in comparison with those of wild type and TR 34 . The nucleotide sequence is targeted for the promoter region for the TR 46 resistance gene (between TR 46 -1 and TR 46 -2). The sequence in this part corresponds to the B2 sequence in Table 2. www.nature.com/scientificreports/ Abs Times Figure 5. Experimental results of the detection limit of the TR 46 LAMP assay. The detection limit reaction was carried out using 10 7 to 10 copies of plasmid DNA per reaction. The detection limit was measured within 60 min. LAMP-method. LAMP was performed as described in our previous studies 23 . TR 46 LAMP primers were designed based on the target promoter region sequences of the cyp51A gene of A. fumigatus, which includes tandem repeats in the promoter region containing TR 46 mutant alleles. The sequence of the cyp51A gene was downloaded from NCBI Gen-Bank (https:// www. ncbi. nlm. nih. gov/ geneb ank, accession numbers AF222068 for wild type, MH231595.1 for TR 34 , and MH040305.1 for TR 46 ). In total, a 184-bp nucleotide alignment ( Fig. 1) was used for TR 46 LAMP primer design by the protocol of the Eiken Company (Primer Explorer V5, Eiken Chemical Co. Ltd, Tokyo. Japan). LAMP primers are composed of six primers recognizing eight distinct regions. LAMP reactions were performed with a Loopamp DNA amplification kit using reaction mixtures composed of 40 pmol each of primers FIP and BIP, 5 pmol each of primers F3 and B3, 20 pmol each of primers LF and LB, 12.5 mL × 2 reaction mixture, 1 μl Bst DNA polymerase, 2 μL DNA sample and distilled water up to a final volume of 25 μL (Eiken Chemical Co., Ltd., Tokyo, Japan). The LAMP reactions were analyzed by a real-time turbidimeter (Loopamp EXIA; Eiken Chemical Co.) and were conducted at 63 °C, for 120 min and then heated at 80 °C for 2 min to terminate the reaction. The start of amplification of LAMP products at 30 to 50 min in the graph suggested the positive reaction due to the presence of corresponding 46 bp tandem repeats of cyp51A gene. Since overall reaction can be obtained within 2 h, prompt drug therapy can be deployed within a short time. To check the detection limit of TR 46 specific LAMP primers, the plasmid DNA was used. To construct plasmid contained TR 46 and cyp51A gene sequences, we cloned the alleles using the shuttle vector pCB1004. Genomic DNA of IFM63432 was used as template to clone the alleles. The cyp51 coding region including approximately 1 kb fragments upstream and downstream were amplified by PCR using the primers pCB1004_Hind_cyp51A-F (5'-aggaattcgatatcaTAG AAT GAG TGA GCT GAT TT-3') and pCB1004_Kpn_cyp51A-R (5'-gggcgaattgggtacCAG GTT TTC GCA CGA GCT TCTCC-3'). Amplified DNA fragments were fused into pCB1004 digested with HindIII and KpnI, by In-Fusion H Cloning Kit (Takara Bio, Otsu, Japan). The size of plasmid DNA was 8164 bp.