eDNA-based monitoring of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans with ddPCR in Luxembourg ponds: taking signals below the Limit of Detection (LOD) into account
Abstract Background Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are two pathogenic fungi that are a significant threat to amphibian communities worldwide. European populations are strongly impacted and the monitoring of the presence and spread of these pathogens...
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| Format: | Article |
| Language: | English |
| Published: |
BMC
2024-01-01
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| Series: | BMC Ecology and Evolution |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12862-023-02189-9 |
| _version_ | 1797363736605884416 |
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| author | David Porco Chanistya Ayu Purnomo Liza Glesener Roland Proess Stéphanie Lippert Kevin Jans Guy Colling Simone Schneider Raf Stassen Alain C. Frantz |
| author_facet | David Porco Chanistya Ayu Purnomo Liza Glesener Roland Proess Stéphanie Lippert Kevin Jans Guy Colling Simone Schneider Raf Stassen Alain C. Frantz |
| author_sort | David Porco |
| collection | DOAJ |
| description | Abstract Background Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are two pathogenic fungi that are a significant threat to amphibian communities worldwide. European populations are strongly impacted and the monitoring of the presence and spread of these pathogens is crucial for efficient decision-making in conservation management. Results Here we proposed an environmental DNA (eDNA) monitoring of these two pathogenic agents through droplet digital PCR (ddPCR) based on water samples from 24 ponds in Luxembourg. In addition, amphibians were swabbed in eight of the targeted ponds in order to compare the two approaches at site-level detection. This study allowed the development of a new method taking below-Limit of Detection (LOD) results into account thanks to the statistical comparison of the frequencies of false positives in no template controls (NTC) and below-LOD results in technical replicates. In the eDNA-based approach, the use of this method led to an increase in Bd and Bsal detection of 28 and 50% respectively. In swabbing, this resulted in 8% more positive results for Bd. In some samples, the use of technical replicates allowed to recover above-LOD signals and increase Bd detection by 35 and 33% respectively for eDNA and swabbing, and Bsal detection by 25% for eDNA. Conclusions These results confirmed the usefulness of technical replicates to overcome high levels of stochasticity in very low concentration samples even for a highly sensitive technique such as ddPCR. In addition, it showed that below-LOD signals could be consistently recovered and the corresponding amplification events assigned either to positive or negative detection via the method developed here. This methodology might be particularly worth pursuing in pathogenic agents’ detection as false negatives could have important adverse consequences. In total, 15 ponds were found positive for Bd and four for Bsal. This study reports the first record of Bsal in Luxembourg. |
| first_indexed | 2024-03-08T16:25:25Z |
| format | Article |
| id | doaj.art-76e46076e004494a8dcb0a11241dacbd |
| institution | Directory Open Access Journal |
| issn | 2730-7182 |
| language | English |
| last_indexed | 2024-03-08T16:25:25Z |
| publishDate | 2024-01-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Ecology and Evolution |
| spelling | doaj.art-76e46076e004494a8dcb0a11241dacbd2024-01-07T12:04:47ZengBMCBMC Ecology and Evolution2730-71822024-01-0124111610.1186/s12862-023-02189-9eDNA-based monitoring of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans with ddPCR in Luxembourg ponds: taking signals below the Limit of Detection (LOD) into accountDavid Porco0Chanistya Ayu Purnomo1Liza Glesener2Roland Proess3Stéphanie Lippert4Kevin Jans5Guy Colling6Simone Schneider7Raf Stassen8Alain C. Frantz9Musée national d’histoire naturelle du LuxembourgMusée national d’histoire naturelle du LuxembourgNaturschutzsyndikat SICONAUmweltplanungsbüro EcotopMusée national d’histoire naturelle du LuxembourgNatur&ëmwelt Fondation Hëllef fir d’NaturMusée national d’histoire naturelle du LuxembourgMusée national d’histoire naturelle du LuxembourgBiota.luMusée national d’histoire naturelle du LuxembourgAbstract Background Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are two pathogenic fungi that are a significant threat to amphibian communities worldwide. European populations are strongly impacted and the monitoring of the presence and spread of these pathogens is crucial for efficient decision-making in conservation management. Results Here we proposed an environmental DNA (eDNA) monitoring of these two pathogenic agents through droplet digital PCR (ddPCR) based on water samples from 24 ponds in Luxembourg. In addition, amphibians were swabbed in eight of the targeted ponds in order to compare the two approaches at site-level detection. This study allowed the development of a new method taking below-Limit of Detection (LOD) results into account thanks to the statistical comparison of the frequencies of false positives in no template controls (NTC) and below-LOD results in technical replicates. In the eDNA-based approach, the use of this method led to an increase in Bd and Bsal detection of 28 and 50% respectively. In swabbing, this resulted in 8% more positive results for Bd. In some samples, the use of technical replicates allowed to recover above-LOD signals and increase Bd detection by 35 and 33% respectively for eDNA and swabbing, and Bsal detection by 25% for eDNA. Conclusions These results confirmed the usefulness of technical replicates to overcome high levels of stochasticity in very low concentration samples even for a highly sensitive technique such as ddPCR. In addition, it showed that below-LOD signals could be consistently recovered and the corresponding amplification events assigned either to positive or negative detection via the method developed here. This methodology might be particularly worth pursuing in pathogenic agents’ detection as false negatives could have important adverse consequences. In total, 15 ponds were found positive for Bd and four for Bsal. This study reports the first record of Bsal in Luxembourg.https://doi.org/10.1186/s12862-023-02189-9Batrachochytrium dendrobatidisBatrachochytrium salamandrivoranseDNABelow-LOD signalddPCRPathogens |
| spellingShingle | David Porco Chanistya Ayu Purnomo Liza Glesener Roland Proess Stéphanie Lippert Kevin Jans Guy Colling Simone Schneider Raf Stassen Alain C. Frantz eDNA-based monitoring of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans with ddPCR in Luxembourg ponds: taking signals below the Limit of Detection (LOD) into account BMC Ecology and Evolution Batrachochytrium dendrobatidis Batrachochytrium salamandrivorans eDNA Below-LOD signal ddPCR Pathogens |
| title | eDNA-based monitoring of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans with ddPCR in Luxembourg ponds: taking signals below the Limit of Detection (LOD) into account |
| title_full | eDNA-based monitoring of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans with ddPCR in Luxembourg ponds: taking signals below the Limit of Detection (LOD) into account |
| title_fullStr | eDNA-based monitoring of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans with ddPCR in Luxembourg ponds: taking signals below the Limit of Detection (LOD) into account |
| title_full_unstemmed | eDNA-based monitoring of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans with ddPCR in Luxembourg ponds: taking signals below the Limit of Detection (LOD) into account |
| title_short | eDNA-based monitoring of Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans with ddPCR in Luxembourg ponds: taking signals below the Limit of Detection (LOD) into account |
| title_sort | edna based monitoring of batrachochytrium dendrobatidis and batrachochytrium salamandrivorans with ddpcr in luxembourg ponds taking signals below the limit of detection lod into account |
| topic | Batrachochytrium dendrobatidis Batrachochytrium salamandrivorans eDNA Below-LOD signal ddPCR Pathogens |
| url | https://doi.org/10.1186/s12862-023-02189-9 |
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