Rapid detection of multiple phytoplasma 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 and Recombinase Polymerase
Amplification (RPA) 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 or less 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 a ideal selection with high specificity
and sensitivity for phytoplasma detection. Our work provides a technique
that can be potentially used to rapidly and simultaneously detect more
pathogens.