Medical Mycology Research Center, Chiba University, Chiba, Chiba, Japan.
Research Institute for Science and Engineering, Waseda University, Tokyo, Japan.
PLoS Negl Trop Dis. 2023 Sep 18;17(9):e0011644. doi: 10.1371/journal.pntd.0011644. eCollection 2023 Sep.
Filamentous fungi of the genus Madurella are the primary causative agents of mycetoma, a disease observed in tropical and subtropical regions. Since early diagnostics based on a morphological approach are difficult and have many shortcomings, a molecular diagnostic method suitable for rural settings is required. In this study, we developed the loop-mediated isothermal amplification (LAMP) method to present a foundational technique of the diagnosis of Madurella spp. (M. mycetomatis, M. pseudomycetomatis, M. tropicana, and M. fahalii), the common causative organisms of eumycetoma.
We successfully designed a primer pair targeting the rDNAs of three Madurella spp. excluding M. fahalii, and detected up to 100 fg of genomic DNA extracted from isolates of M. mycetomatis and 1 pg of M. pseudomycetomatis and M. tropicana, within one hour. Second, a primer pair specific to M. mycetomatis, the most common causative species, or M. fahalii, a drug-resistant species, was constructed, and the detection limit of both primer pairs was 1 pg. The designed primers accurately distinguished 16 strains of the genus Madurella from various fungal species known to cause mycetomas.
In summary, we established the first model of a LAMP detection method that rapidly and sensitively detects and identifies Madurella isolates for clinical diagnostics. Moreover, the combined designed primer sets could identify mycetoma-causing strains simultaneously.
马杜拉真菌属的丝状真菌是足菌肿的主要病原体，这种疾病发生在热带和亚热带地区。由于基于形态学方法的早期诊断既困难又有许多缺点，因此需要一种适合农村环境的分子诊断方法。在这项研究中，我们开发了环介导等温扩增（LAMP）方法，为马杜拉菌属（M. mycetomatis、M. pseudomycetomatis、M. tropicana 和 M. fahalii）的诊断提供了基础技术，马杜拉菌属是真菌性足菌肿的常见病原体。
我们成功设计了一对针对三种马杜拉菌属（不包括 M. fahalii）rDNA 的引物，在一小时内可检测到从 M. mycetomatis 分离株中提取的高达 100 fg 基因组 DNA 和 1 pg 的 M. pseudomycetomatis 和 M. tropicana。其次，构建了一对特异性针对最常见病原体 M. mycetomatis 或耐药病原体 M. fahalii 的引物，这两个引物对的检测限均为 1 pg。设计的引物能够准确地区分来自各种已知引起足菌肿的真菌物种的 16 株马杜拉菌属。
总之，我们建立了第一个快速灵敏检测和鉴定临床诊断中马杜拉菌属分离株的 LAMP 检测方法模型。此外，组合设计的引物组可以同时鉴定引起足菌肿的菌株。
