Rapid detection of multiple phytoplasmas with an All-In-One Dual (AIOD) CRISPR assay

Phytoplasma can infect thousands of plants and caused huge economic losses around the world. The large-scale spread and serious lethality of phytoplasma prompt the urgent need for sensitive, accurate, visual and rapid detection of these pathogens. Current molecular assays used for detecting phytoplasma are expensive and time consuming. Here, we established a novel All-In-One Dual (AIOD) CRISPR detection platform based on CRISPR/LbCas12a technology for the diagnosis of phytoplasmas. The protocol is simple, requiring one vessel, rapid and sensitive, and the output is visual. Phytoplasmas can be detected after 15 min from leaf harvest with this platform. In the optimized reaction, the concentration of ssDNA-FQ, LbCas12a, crRNA1 and crRNA2 were 1,000 nM, 1.6 μM, 0.8μM and 0.8 μM, respectively. Our AIOD-CRISPR detection system reach the sensitive threshold of 3.37 E+2 copies of phytoplasma DNA per reaction. Field tests indicated the AIOD-CRISPR detection system possessed high specificity and reached the 100% accuracy when compared with PCR detection. In conclusion, the AIOD-CRISPR detection platform is more convenient and efficient than traditional phytoplasma detection methods and possesses great potential in field detection application.