Development of a recombinase-aided amplification coupled with lateral flow dipstick assay for tobacco Corynespora leaf spot disease detection

Tobacco Corynespora leaf spot disease caused by Corynespora cassiicola seriously affects the quality of flue-cured tobacco. However, current detection assays are not able to quickly and reliably identify infected leaves from multiple similar diseases. In this study, an assay consisting of recombinase-aided amplification combined with lateral-flow dipstick technology (RAA-LFD) was developed for detecting tobacco Corynespora leaf spot disease. Under constant temperature conditions of 37 ℃, DNA amplification can be completed within 20 minutes. The assay could accurately diagnose the C. cassiicola among 12 tobacco pathogens. Sensitivity of the RAA-LFD assay was tested by diluting DNA from 100 ng to 1 pg with a 10-fold gradient, and the results showed that the sensitivity of the assay was 10 pg, which is comparable to conventional PCR assay. In addition, combined with the crude extraction of DNA from field disease samples using Alkaline PEG lysis buffer, the detection could be completed within 30 minutes. Our results indicated that the RAA-LFD assay would be rapid, sensitive, and specific for the detection of tobacco Corynespora leaf spot disease.