Frontiers in Bioengineering and Biotechnology (Jun 2022)
CRISPR/Cas12a Coupled With Recombinase Polymerase Amplification for Sensitive and Specific Detection of Aphelenchoides besseyi
Abstract
Aphelenchoides besseyi (A. besseyi), a seed-borne parasitic nematode, is the causal agent of rice white tip disease (RWTD), which may result in a drastic loss of rice yield. Seed treatments are currently considered to be the most effective means of preventing the spread of RWTD. Therefore, the rapid, highly specific, and accurate detection of A. besseyi from rice seeds is crucial for the surveillance, prevention, and control of RWTD. Here, we describe a novel detection assay that combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a to detect A. besseyi (termed RPA-Cas12a-Ab), with a low limit of detection (LOD) of 1 copy/μl of plasmid or 1:107 diluted DNA extracted from individual nematodes. To improve the user-friendliness, lateral flow strip assay (LFA) was adopted to visualize the detection result. The LOD of the RPA-Cas12a-Ab LFA assay was 1,000 copies/μl plasmid or 1:10 diluted DNA extracted from individual nematodes. The assay developed in this study was able to identify A. besseyi in 45 min with high accuracy and sensitivity without cross reaction with three closely related non-A. besseyi species. Thus, RPA-Cas12a-Ab is a rapid, sensitive, and specific detection system that requires no sophisticated equipment and shows promise for on-site surveillance of A. besseyi.
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