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

Trabasso, Plı́nio; Matsuzawa, T.; Arai, Teppei; Hagiwara, Daisuke; Mikami, Yuzuru; Moretti, Maria Luíza; Watanabe, Akira

doi:10.1038/s41598-021-96651-7

Cited by 10 publications

(4 citation statements)

References 24 publications

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“…within 60 min, as shown in Fig 4 . Based on the estimated molecular number of genomic DNA of M. mycetomatis, which is calculated to be 26.6 copies/pg, assuming a genome size of 36.7 Mbp [26]. The system detection limits are comparable not only to those targeting other fungi, such as Aspergillus [27] and Fusarium [28] but also to those of real-time PCR methods for Madurella (up to 3 pg) [16]. Furthermore, the sensitivity of our LAMP system is approximately 200 times higher than that reported previously [18].…”

Section: Discussionmentioning

confidence: 99%

Specific and sensitive loop-mediated isothermal amplification (LAMP) method for Madurella strains, eumycetoma filamentous fungi causative agent

Yoshioka,

Mori,

Fahal

et al. 2023

PLoS Negl Trop Dis

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Background 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. Principal findings 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. Conclusion 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.

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Section: Discussionmentioning

confidence: 99%

Specific and sensitive loop-mediated isothermal amplification (LAMP) method for Madurella strains, eumycetoma filamentous fungi causative agent

Yoshioka,

Mori,

Fahal

et al. 2023

PLoS Negl Trop Dis

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“…Other methods have been developed for rapid detection of A. fumigatus, including realtime PCR [20] and loop-mediated isothermal amplification (LAMP) [34,35]. These methods have been applied to clinical samples or to isolates in the case of LAMP.…”

Section: Plos Onementioning

confidence: 99%

“…Nevertheless, the development of a real-time PCR-based method for the direct detection and quantification of resistant A. fumigatus from environmental samples would be very useful. LAMP is a practical and easy method for rapid detection but the currently developed methods [34,35] have not been tested on environmental samples. Moreover, in a single assay the nested PCR assays discriminates wt, TR34 and TR46 alleles, which are the two alleles most commonly associated with the usage of azoles in the agriculture [11].…”

Section: Plos Onementioning

confidence: 99%

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Detection of azole-resistant Aspergillus fumigatus in the environment from air, plant debris, compost, and soil

Londoño

Brewer²

2023

PLoS ONE

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Aspergillus fumigatus is a ubiquitous fungus, a saprophyte of plants, and an opportunistic pathogen of humans. Azole fungicides are used in agriculture to control plant pathogens, and azoles are also used as a first line of treatment for aspergillosis. The continued exposure of A. fumigatus to azoles in the environment has likely led to azole resistance in the clinic where infections result in high levels of mortality. Pan-azole resistance in environmental isolates is most often associated with tandem-repeat (TR) mutations containing 34 or 46 nucleotides in the cyp51A gene. Because the rapid detection of resistance is important for public health, PCR-based techniques have been developed to detect TR mutations in clinical samples. We are interested in identifying agricultural environments conducive to resistance development, but environmental surveillance of resistance has focused on labor-intensive isolation of the fungus followed by screening for resistance. Our goal was to develop assays for the rapid detection of pan-azole-resistant A. fumigatus directly from air, plants, compost, and soil samples. To accomplish this, we optimized DNA extractions for air filters, soil, compost, and plant debris and standardized two nested-PCR assays targeting the TR mutations. Sensitivity and specificity of the assays were tested using A. fumigatus DNA from wild type and TR-based resistant isolates and with soil and air filters spiked with conidia of the same isolates. The nested-PCR assays were sensitive to 5 fg and specific to A. fumigatus without cross-reaction with DNA from other soil microorganisms. Environmental samples from agricultural settings in Georgia, USA were sampled and tested. The TR46 allele was recovered from 30% of samples, including air, soil and plant debris samples from compost, hibiscus and hemp. These assays allow rapid surveillance of resistant isolates directly from environmental samples improving our identification of hotspots of azole-resistant A. fumigatus.

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Rapid and quantitative detection of Aspergillus niger Van Tieghem using loop-mediated isothermal amplification assay

Dai,

Cao,

et al. 2024

J Plant Pathol

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Peanut (Arachis hypogaea L.) crown rot and root rot are common diseases caused by Aspergillus niger Van Tieghem. Early and accurate detection of A. niger is key to disease management. In this study, the design of two to five sets of loop-mediated isothermal amplification (LAMP) primers was based on the EglA, GOD, Tub, NRPS, Tan, CbhA, and CbhB genes of A. niger. Of these, primer set GOD-91 was selected for optimization of the three-factor LAMP system: the Bst DNA polymerase concentration, the concentration ratio of the inner and outer primers, and the concentration of Mg2+. In addition, the optimized LAMP reaction system for A. niger detection was validated for specificity, sensitivity, and on-site feasibility. The specificity test showed that A. niger could be specifically detected with the proposed method without cross-amplification of other pathogenic fungi DNA. Moreover, based on the sensitivity test, the lowest detection limit of this reaction system was 5.1 × 10−7 ng/µL pAN01 plasmid DNA, after which a standard curve was generated for the quantitative detection of A. niger. The LAMP method was further applied for field sample assessment before and after A. niger infection, successfully detecting A. niger presence in the samples collected in the field. This study yielded a sensitive, specific, and reproducible LAMP system that can be used to assess on-site samples within 45 min. It is an effective approach for the rapid and quantitative detection of A. niger.

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

Cited by 10 publications

References 24 publications

Specific and sensitive loop-mediated isothermal amplification (LAMP) method for Madurella strains, eumycetoma filamentous fungi causative agent

Specific and sensitive loop-mediated isothermal amplification (LAMP) method for Madurella strains, eumycetoma filamentous fungi causative agent

Detection of azole-resistant Aspergillus fumigatus in the environment from air, plant debris, compost, and soil

Rapid and quantitative detection of Aspergillus niger Van Tieghem using loop-mediated isothermal amplification assay

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