The primers’ design specific to <i>M</i>. <i>mycetomatis</i> (primer PM) or <i>M</i>. <i>fahalii</i> (primer PF).

The rDNA sequence of M. mycetomatis (A) or M. fahalii (B) was aligned with that of M. pseudomycetomatis (accession no. EU815933.1), and arrows represented the primer positions. The matched nucleotide position was represented using dot signs, while the gap and mismatch positions using corresponding substitution ones. The species name and the number of first nucleotide were represented at each line using the following abbreviations: MM, M. mycetomatis; MP, M. pseudomycetomatis; MF, M. fahalii. The nucleotide alignment and illustration were performed using Genetyx Ver. 14 (Genetyx Co., Tokyo, Japan) with its default parameters.</p

Photograph showing recurrence at the right below-knee stump.

Photograph showing recurrence at the right below-knee stump.</p

Chest CT showing segmental/lobar pneumonia with parapneumonic pleural effusion.

Chest CT showing segmental/lobar pneumonia with parapneumonic pleural effusion.</p

LAMP amplification for clinical isolates and pathogenic fungi and bacteria, which are known as causative agents of mycetoma.

LAMP amplification for clinical isolates and pathogenic fungi and bacteria, which are known as causative agents of mycetoma.</p

Design of primer targeting three <i>Madurella</i> spp. (primer PV).

The alignment of rDNA sequences among Madurella strains and Chaeromium rectangluare (negative control) is shown. Arrows illustrate the primers’ positions. The species name and the number of the first nucleotide are represented at each line using the abbreviations as followings: MM, M. mycetomatis (accession no. DQ836767.1); MP, M. pseudomycetomatis (accession no. EU815933.1); MT, M. tropicana (accession no. MK926825.1); MF, M. fahalii (accession no. JN573178.1); CR, Chaetomium rectangluare (NR_144817). The nucleotide alignment and illustration are performed using Genetyx Ver. 14 (Genetyx Co., Tokyo, Japan) with its default parameters.</p

Verification of LAMP amplicons using primer sets PM, PM and PF by agarose electrophoresis.

(A) PM (Lanes 1–9) and (B) PM (Lanes 10–13) and PF (Lanes 14–17) by agarose electrophoresis. Lanes: M, EZ load 100 bp Molecular Ruler (Bio-rad, Hercules, CA); 1, Madurella mycetomatis IFM 46458 (10 ng); 2, M. pseudomycetomatis IFM 46460 (10 ng); 3, M. tropicana CBS 201.38 (10 ng); 4, M. fahalii CBS 129176 (10 ng); 5, Chaetomium rectangulare CBS 126778 (10 ng); 6, M. mycetomatis IFM 46458 (1 pg); 7, M. pseudomycetomatis IFM 46460 (1 pg); 8, M. tropicana CBS 201.38 (1 pg); 9, no template; 10, M. mycetomatis IFM 46458 (10 ng); 11, M. mycetomatis IFM 46458 (1 pg); 12, M. fahalii CBS 129176 (10 ng); 13, no template; 14, M. fahalii CBS 129176 (10 ng); 15, M. fahalii CBS 129176 (1 pg); 16, M. mycetomatis IFM 46458 (10 ng); 17, no template. (TIF)</p

Fig 3 -

The specificity of designed LAMP primers, (A) PV, (B) PM and (C) PF. 10 ng of the extracted genomic DNAs derived from Madurella spp. were used as templates. LAMP reaction was performed at 63°C for 60 min, and the turbidity of the reaction solution was monitored as an indicator of amplification. As for primer PV, the genomic DNA of Chaetomium rectangulare was also used. The sample legends of the plots were represented on the bottom right panel. Abbreviations: MM, M. mycetomatis IFM 46458; MP, M. pseudomycetomatis IFM 46460; MT, M. tropicana CBS201.38; MF, M. fahalii CBS129176; CR, Chaetomium rectangluare CBS 126778; B, Blank sample with a buffer used for DNA dilution.</p

Optimization of LAMP reaction temperature using primer PF.

LAMP reaction was performed using genomic DNA of Madurella fahalii CBS 129176 at the reaction temperature between 62°C and 65°C. The solid line and dotted line represent the data of the genomic DNA sample and blank using a buffer used for DNA dilution, respectively. (TIF)</p

Optimization of LAMP reaction temperature using primer PM.

LAMP reaction was performed using genomic DNA of Madurella myceromatis IFM 46458 at the reaction temperature between 62°C and 65°C. The solid line and dotted line represent the data of the genomic DNA sample and blank using a buffer used for DNA dilution, respectively. (TIF)</p

The primers’ design targeting all four Madurella spp. (primer PV2).

(A) The alignment of rDNA sequences among Madurella strains and Chaeromium rectangluare (negative control) is shown. Arrows illustrate the primers’ positions. (B) The specificity of primer PV2. LAMP reaction was performed using genomic DNAs derived from Madurella spp. and C. rectangulare at 65°C. Abbreviations: MM, M. mycetomatis; MP, M. pseudomycetomatis; MT, M. tropicana; MF, M. fahalii; CR, Chaetomium rectangluare; B, Blank sample with a buffer used for DNA dilution. (TIF)</p