FINDeM: A CRISPR-based, molecular method for rapid, inexpensive and field-deployable organism detection

DOI

10.1111/2041-210X.14236

Document Type

Journal Article

Publication Date

1-1-2023

Publication Title

Methods in Ecology and Evolution

Keywords

Batrachochytrium dendrobatidis, CRISPR-Cas12a, DETECTR, field-based, guide RNA, isothermal, rapid DNA extraction, recombinase polymerase amplification

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.

Open Access

Gold

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