FINDeM: A CRISPR‐based, molecular method for rapid, inexpensive and field‐deployable organism detection
Abstract The field of ecology has undergone a molecular revolution, with researchers increasingly relying on DNA‐based methods for organism detection. Unfortunately, these techniques often require expensive equipment, dedicated laboratory spaces and specialized training in molecular and computationa...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Wiley
2023-12-01
|
| Series: | Methods in Ecology and Evolution |
| Subjects: | |
| Online Access: | https://doi.org/10.1111/2041-210X.14236 |
| _version_ | 1797403234451587072 |
|---|---|
| author | Brandon D. Hoenig Jakub Zegar Michel E. B. Ohmer Macie M. Chess Brady A. Porter Myah Madril Corinne L. Richards‐Zawacki |
| author_facet | Brandon D. Hoenig Jakub Zegar Michel E. B. Ohmer Macie M. Chess Brady A. Porter Myah Madril Corinne L. Richards‐Zawacki |
| author_sort | Brandon D. Hoenig |
| collection | DOAJ |
| description | Abstract The field of ecology has undergone a molecular revolution, with researchers increasingly relying on DNA‐based methods for organism detection. Unfortunately, these techniques often require expensive equipment, dedicated laboratory spaces and specialized training in molecular and computational techniques; limitations that may exclude field researchers, underfunded programmes and citizen scientists from contributing to cutting‐edge science. It is for these reasons that we have designed a simplified, inexpensive method for field‐based molecular organism detection—FINDeM (Field‐deployable Isothermal Nucleotide‐based Detection Method). In this approach, DNA is extracted using chemical cell lysis and a cellulose filter disc, followed by two body‐heat inducible reactions—recombinase polymerase amplification and a CRISPR‐Cas12a fluorescent reporter assay—to amplify and detect target DNA, respectively. Here, we introduce and validate FINDeM in detecting Batrachochytrium dendrobatidis, the causative agent of amphibian chytridiomycosis, and show that this approach can identify single‐digit DNA copies from epidermal swabs in under 1 h using low‐cost supplies and field‐friendly equipment. This research signifies a breakthrough in ecology, as we demonstrate a field‐deployable platform that requires only basic supplies (i.e. micropipettes, plastic consumables and a UV flashlight), inexpensive reagents (~$1.29 USD/sample) and emanated body heat for highly sensitive, DNA‐based organism detection. By presenting FINDeM in an ecological system with pressing, global biodiversity implications, we aim to not only highlight how CRISPR‐based applications promise to revolutionize organism detection but also how the continued development of such techniques will allow for additional, more diversely trained researchers to answer the most pressing questions in ecology. |
| first_indexed | 2024-03-09T02:35:37Z |
| format | Article |
| id | doaj.art-903e645768454347af059b4128bae12c |
| institution | Directory Open Access Journal |
| issn | 2041-210X |
| language | English |
| last_indexed | 2024-03-09T02:35:37Z |
| publishDate | 2023-12-01 |
| publisher | Wiley |
| record_format | Article |
| series | Methods in Ecology and Evolution |
| spelling | doaj.art-903e645768454347af059b4128bae12c2023-12-06T10:20:16ZengWileyMethods in Ecology and Evolution2041-210X2023-12-0114123055306710.1111/2041-210X.14236FINDeM: A CRISPR‐based, molecular method for rapid, inexpensive and field‐deployable organism detectionBrandon D. Hoenig0Jakub Zegar1Michel E. B. Ohmer2Macie M. Chess3Brady A. Porter4Myah Madril5Corinne L. Richards‐Zawacki6Department of Biological Sciences University of Pittsburgh Pittsburgh Pennsylvania USADepartment of Biology The University of Mississippi University Park Mississippi USADepartment of Biology The University of Mississippi University Park Mississippi USADepartment of Biological Sciences Duquesne University Pittsburgh Pennsylvania USADepartment of Biological Sciences Duquesne University Pittsburgh Pennsylvania USADepartment of Biological Sciences University of Pittsburgh Pittsburgh Pennsylvania USADepartment of Biological Sciences University of Pittsburgh Pittsburgh Pennsylvania USAAbstract The field of ecology has undergone a molecular revolution, with researchers increasingly relying on DNA‐based methods for organism detection. Unfortunately, these techniques often require expensive equipment, dedicated laboratory spaces and specialized training in molecular and computational techniques; limitations that may exclude field researchers, underfunded programmes and citizen scientists from contributing to cutting‐edge science. It is for these reasons that we have designed a simplified, inexpensive method for field‐based molecular organism detection—FINDeM (Field‐deployable Isothermal Nucleotide‐based Detection Method). In this approach, DNA is extracted using chemical cell lysis and a cellulose filter disc, followed by two body‐heat inducible reactions—recombinase polymerase amplification and a CRISPR‐Cas12a fluorescent reporter assay—to amplify and detect target DNA, respectively. Here, we introduce and validate FINDeM in detecting Batrachochytrium dendrobatidis, the causative agent of amphibian chytridiomycosis, and show that this approach can identify single‐digit DNA copies from epidermal swabs in under 1 h using low‐cost supplies and field‐friendly equipment. This research signifies a breakthrough in ecology, as we demonstrate a field‐deployable platform that requires only basic supplies (i.e. micropipettes, plastic consumables and a UV flashlight), inexpensive reagents (~$1.29 USD/sample) and emanated body heat for highly sensitive, DNA‐based organism detection. By presenting FINDeM in an ecological system with pressing, global biodiversity implications, we aim to not only highlight how CRISPR‐based applications promise to revolutionize organism detection but also how the continued development of such techniques will allow for additional, more diversely trained researchers to answer the most pressing questions in ecology.https://doi.org/10.1111/2041-210X.14236Batrachochytrium dendrobatidisCRISPR‐Cas12aDETECTRfield‐basedguide RNAisothermal |
| spellingShingle | Brandon D. Hoenig Jakub Zegar Michel E. B. Ohmer Macie M. Chess Brady A. Porter Myah Madril Corinne L. Richards‐Zawacki FINDeM: A CRISPR‐based, molecular method for rapid, inexpensive and field‐deployable organism detection Methods in Ecology and Evolution Batrachochytrium dendrobatidis CRISPR‐Cas12a DETECTR field‐based guide RNA isothermal |
| title | FINDeM: A CRISPR‐based, molecular method for rapid, inexpensive and field‐deployable organism detection |
| title_full | FINDeM: A CRISPR‐based, molecular method for rapid, inexpensive and field‐deployable organism detection |
| title_fullStr | FINDeM: A CRISPR‐based, molecular method for rapid, inexpensive and field‐deployable organism detection |
| title_full_unstemmed | FINDeM: A CRISPR‐based, molecular method for rapid, inexpensive and field‐deployable organism detection |
| title_short | FINDeM: A CRISPR‐based, molecular method for rapid, inexpensive and field‐deployable organism detection |
| title_sort | findem a crispr based molecular method for rapid inexpensive and field deployable organism detection |
| topic | Batrachochytrium dendrobatidis CRISPR‐Cas12a DETECTR field‐based guide RNA isothermal |
| url | https://doi.org/10.1111/2041-210X.14236 |
| work_keys_str_mv | AT brandondhoenig findemacrisprbasedmolecularmethodforrapidinexpensiveandfielddeployableorganismdetection AT jakubzegar findemacrisprbasedmolecularmethodforrapidinexpensiveandfielddeployableorganismdetection AT michelebohmer findemacrisprbasedmolecularmethodforrapidinexpensiveandfielddeployableorganismdetection AT maciemchess findemacrisprbasedmolecularmethodforrapidinexpensiveandfielddeployableorganismdetection AT bradyaporter findemacrisprbasedmolecularmethodforrapidinexpensiveandfielddeployableorganismdetection AT myahmadril findemacrisprbasedmolecularmethodforrapidinexpensiveandfielddeployableorganismdetection AT corinnelrichardszawacki findemacrisprbasedmolecularmethodforrapidinexpensiveandfielddeployableorganismdetection |