Comparative Evaluation of PCR-Based, LAMP and RPA-CRISPR/Cas12a Assays for the Rapid Detection of Diaporthe aspalathi.

Abstract

Southern stem canker (SSC) of soybean, attributable to the fungal pathogen Diaporthe aspalathi, results in considerable losses of soybean in the field and has damaged production in several of the main soybean-producing countries worldwide. Early and precise identification of the causal pathogen is imperative for effective disease management. In this study, we performed an RPA-CRISPR/Cas12a, as well as LAMP, PCR and real-time PCR assays to verify and compare their sensitivity, specificity and simplicity and the practicality of the reactions. We screened crRNAs targeting a specific single-copy gene, and optimized the reagent concentrations, incubation temperatures and times for the conventional PCR, real-time PCR, LAMP, RPA and Cas12a cleavage stages for the detection of D. aspalathi. In comparison with the PCR-based assays, two thermostatic detection technologies, LAMP and RPA-CRISPR/Cas12a, led to higher specificity and sensitivity. The sensitivity of the LAMP assay could reach 0.01 ng μL-1 genomic DNA, and was 10 times more sensitive than real-time PCR (0.1 ng μL-1) and 100 times more sensitive than conventional PCR assay (1.0 ng μL-1); the reaction was completed within 1 h. The sensitivity of the RPA-CRISPR/Cas12a assay reached 0.1 ng μL-1 genomic DNA, and was 10 times more sensitive than conventional PCR (1.0 ng μL-1), with a 30 min reaction time. Furthermore, the feasibility of the two thermostatic methods was validated using infected soybean leaf and seeding samples. The rapid, visual one-pot detection assay developed could be operated by non-expert personnel without specialized equipment. This study provides a valuable diagnostic platform for the on-site detection of SSC or for use in resource-limited areas.