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

Abstract

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 multiple phytoplasma. The protocol is simple, requiring one vessel, rapid and sensitive, and the output is visual. Cas12a/crRNAs complexes are added into a reaction containing RPA Mix, RPA primers and single-stranded DNA fluorophore-quencher (ssDNA-FQ). All components, including 1 μL of sample DNA, are added together and then incubated in one tube at 37 °C. Phytoplasma was detected after 15 min from leaf harvest. Positive results can be observed by the naked eye via fluorescent signals. We optimized the amounts of crRNA, LbCas12a and the ssDNA fluorophore in the detection system. Finally, an optimized system was established containing 1,000 nM ssDNA-FQ and a 2:1:1 ratio of LbCas12a/crRNA1/crRNA2 complex with a 0.8 μM concentration as 1. In the optimized reaction, the AIOD-CRISPR detection system exhibited high sensitivity, with limits of detection reaching 3.37E + 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 system is an ideal selection with high specificity and sensitivity for phytoplasma detection.