Environmental Screening of Fonsecaea Agents of Chromoblastomycosis Using Rolling Circle Amplification
Abstract
:1. Introduction
2. Materials and Methods
2.1. Study Area and Samples
2.2. Padlock Probes
2.2.1. DNA Extraction of Environmental Samples
2.2.2. DNA Amplification
2.2.3. Ligation of Padlock Probes
2.2.4. Rolling Circle Amplification Reaction
2.2.5. Specificity and Detection Limit of RCA Padlock Probes In Vitro and In Vivo
2.3. Isolation and Molecular Identification
2.3.1. Isolation
2.3.2. Molecular Identification
3. Results
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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DNA Test | Species | Collection Number | Source/Geography | Padlock Probe | |
---|---|---|---|---|---|
FOP | FOM | ||||
Fungal DNA | F. pedrosoi | CBS271.37 T | Chromoblastomycosis, South America | (+) | (−) |
F. monophora | CBS269.37 T | Chromoblastomycosis, South America | (−) | (+) | |
F. erecta | CBS125763 T | Spine of Japecanga plant, Brazil, Bacabeira | (−) | (−) | |
F. nubica | CBS125.198 T | Chromoblastomycosis, Cameroon | (−) | (−) | |
F. pugnacious | CMRP1343 T | Chromoblastomycosis, South America | (−) | (−) | |
C. albicans | CMRP816 | Human, Brazil | (−) | (−) | |
P. citrinum | CMRP1538 | Metal, Tucuruí, Brazil | (−) | (−) | |
A. nidulans | CMRP2338 | - | (−) | (−) | |
Environmental test DNA samples | M. pudica | Plant in vitro | (−) | (−) | |
M. pudica with F. pedrosoi | CBS271.37 T | Plant in vitro (20 ng/µL) with 0.5 ng/µL of F. pedrosoi | (+) | (−) | |
M. pudica with F. monophora | CBS269.37 T | Plant in vitro (20 ng/µL) with 0.5 ng/µL of F. monophora | (−) | (+) | |
M. pudica with F. erecta | CBS125763 T | Plant in vitro (20 ng/µL) with 0.5 ng/µL of F. erecta | (−) | (−) | |
B. gasipaes with F. pedrosoi | CBS271.37 T | Plant inoculated with 105 spores of F. pedrosoi | (+) | (−) | |
B. gasipaes with F. monophora | CBS269.37 T | Plant inoculated with 105 spores of F. monophora | (−) | (+) | |
B. gasipaes with F. erecta | CBS125763 T | Plant inoculated with 105 spores of F. erecta | (−) | (−) |
Positive Sample | Substrate | Padlock Probe Positive | N. Isolates | CMRP | Molecular ID. | GenBank Accession |
---|---|---|---|---|---|---|
1 | Decomposing Material | FOP; FOM | 4 | CMRP2566 | Melanoctona tectonae | MT075634 |
CMRP2821 | Melanoctona tectonae | MT075635 | ||||
CMRP2840 | Cladosporium sp. | MT075636 | ||||
CMRP2863 | Cyphellophora sp. | MT075637 | ||||
2 | Decomposing Material | FOP; FOM | 2 | CMRP2617 | Strelitziana sp. | MT080291 |
CMRP2859 | Cyphellophora ambigua | MT075638 | ||||
4 | Decomposing Material | FOM | 5 | CMRP2619 | Cladosporium sp. | MT075639 |
CMRP2594 | Cladosporium sp. | MT075640 | ||||
CMRP2598 | Mycosphaerellaceae | MT080292 | ||||
CMRP2826 | Exophiala alcalophila | MT075641 | ||||
CMRP2822 | Exophiala spinifera | MT075642 | ||||
6 | Leaf, A. vulgare | FOM | 2 | CMRP2601 | Hyalocladosporiella cannae | MT075643 |
CMRP2850 | Exophiala spinifera | MT075644 | ||||
7 | Leaf, S. paniculatum | FOP; FOM | 14 | CMRP2560 | Hyalocladosporiella cannae | MT075645 |
CMRP2848 | Hyalocladosporiella cannae | MT075646 | ||||
CMRP2564 | Hyalocladosporiella cannae | MT075647 | ||||
CMRP2567 | Hyalocladosporiella cannae | MT075648 | ||||
CMRP2557 | Hyalocladosporiella cannae | MT075649 | ||||
CMRP2589 | Nigrograna obliqua | MT075650 | ||||
CMRP2591 | Hyalocladosporiella cannae | MT075651 | ||||
CMRP2609 | Hyalocladosporiella cannae | MT075652 | ||||
CMRP2615 | Hyalocladosporiella cannae | MT075653 | ||||
CMRP2851 | Hyalocladosporiella cannae | MT075654 | ||||
CMRP2852 | Hyalocladosporiella cannae | MT075655 | ||||
CMRP3098 | Hyalocladosporiella cannae | MT075656 | ||||
CMRP3094 | Hyalocladosporiella cannae | MT075657 | ||||
CMRP2867 | Hyalocladosporiella cannae | MT075658 | ||||
8 | Leaf, S. dulcis | FOM | 1 | CMRP2868 | Hyalocladosporiella cannae | MT075659 |
17 | Stalk, S. paniculatum | FOM | 11 | CMRP2562 | Chaetothyriales | MT080293 |
CMRP2569 | Cyphellophora sp. | MT075660 | ||||
CMRP2620 | Cladosporium sp. | MT075661 | ||||
CMRP2568 | Hyalocladosporiella cannae | MT075662 | ||||
CMRP2586 | Hyalocladosporiella cannae | MT075663 | ||||
CMRP2622 | Mycosphaerellaceae | MT080294 | ||||
CMRP2614 | Hyalocladosporiella cannae | MT075664 | ||||
CMRP2828 | Strelitziana sp. | MT080295 | ||||
CMRP2837 | Cyphellophora oxyspora | MT075665 | ||||
CMRP2839 | Teratosphaeria sp. | MT080296 | ||||
CMRP3086 | Chaetothyriales | MT080297 | ||||
35 | Stalk, A. vulgare | FOM | 1 | CMRP2624 | Strelitziana sp. | MT080298 |
47 | Leaf, S. dulcis | FOM | 1 | CMRP3082 | Mycosphaerellaceae | MT080299 |
51 | Stalk, S. dulcis | FOM | 6 | CMRP3116 | Cladosporium sp. | MT075666 |
CMRP3114 | Ochroconis sp. | MT075667 | ||||
CMRP3113 | Cladosporium sp. | MT075668 | ||||
CMRP3001 | Pyriculariaceae | MT080300 | ||||
CMRP3074 | Chaetothyriales | MT080301 | ||||
CMRP2985 | Mycosphaerellaceae | MT080302 | ||||
57 | Decomposing Material | FOM | 6 | CMRP2986 | Mycosphaerellaceae | MT080303 |
CMRP2998 | Fonsecaea brasiliensis | MT075669 | ||||
CMRP3104 | Mycosphaerellaceae | MT080304 | ||||
CMRP3085 | Mycosphaerellaceae | MT080305 | ||||
CMRP3107 | Mycosphaerellaceae | MT080306 | ||||
CMRP3088 | Chaetothyriales | MT080307 | ||||
59 | Decomposing Material | FOM | 8 | CMRP2855 | Chaetothyriales | MT080308 |
CMRP2856 | Chaetothyriales | MT080309 | ||||
CMRP2865 | Chaetothyriales | MT080310 | ||||
CMRP2869 | Chaetothyriales | MT080311 | ||||
CMRP2874 | Chaetothyriales | MT080312 | ||||
CMRP3002 | Chaetothyriales | MT080313 | ||||
CMRP3109 | Fonsecaea brasiliensis | MT075670 | ||||
CMRP2582 | Cladosporium sp. | MT075671 | ||||
71 | Soil | FOM | 1 | CMRP2561 | Exophiala spinifera | MT075672 |
72 | Soil | FOM | 5 | CMRP2580 | Mycosphaerellaceae | MT080314 |
CMRP2602 | Exophiala spinifera | MT075673 | ||||
CMRP2605 | Exophiala spinifera | MT075674 | ||||
CMRP2610 | Trichomeriaceae | MT080315 | ||||
CMRP3117 | Cyphellophora oxyspora | MT075675 | ||||
Total | 67 |
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Voidaleski, M.F.; Gomes, R.R.; Azevedo, C.d.M.P.e.S.d.; Lima, B.J.F.d.S.; Costa, F.d.F.; Bombassaro, A.; Fornari, G.; Cristina Lopes da Silva, I.; Andrade, L.V.; Lustosa, B.P.R.; et al. Environmental Screening of Fonsecaea Agents of Chromoblastomycosis Using Rolling Circle Amplification. J. Fungi 2020, 6, 290. https://doi.org/10.3390/jof6040290
Voidaleski MF, Gomes RR, Azevedo CdMPeSd, Lima BJFdS, Costa FdF, Bombassaro A, Fornari G, Cristina Lopes da Silva I, Andrade LV, Lustosa BPR, et al. Environmental Screening of Fonsecaea Agents of Chromoblastomycosis Using Rolling Circle Amplification. Journal of Fungi. 2020; 6(4):290. https://doi.org/10.3390/jof6040290
Chicago/Turabian StyleVoidaleski, Morgana Ferreira, Renata Rodrigues Gomes, Conceição de Maria Pedrozo e Silva de Azevedo, Bruna Jacomel Favoreto de Souza Lima, Flávia de Fátima Costa, Amanda Bombassaro, Gheniffer Fornari, Isabelle Cristina Lopes da Silva, Lucas Vicente Andrade, Bruno Paulo Rodrigues Lustosa, and et al. 2020. "Environmental Screening of Fonsecaea Agents of Chromoblastomycosis Using Rolling Circle Amplification" Journal of Fungi 6, no. 4: 290. https://doi.org/10.3390/jof6040290