Establishment of RT-RAA-CRISPR/Cas12a Based Visual Detection Method for Pepper Mild Mottle Virus
Pepper is an important horticultural crop.Pepper mild mottle virus(PMMoV)threatens pepper and other solanaceous crops'production.In order to improve the prevention and control efficiency of PMMoV,this study designed specific RAA primers based on the conserved sequence of its gene encoding coat protein(CP)to achieve rapid isothermal amplification of PMMoV based on recombinase-aided amplification(RAA).And crRNA was designed to target the RT-RAA amplification products based on the CRISPR/Cas12a system.The RT-RAA-CRISPR/Cas12a system was optimized,and the results showed that the strongest signal was observed in the total system at a final concentration of 400 nmol/L for reporter gene FQ,and a ratio of 1∶5 for Cas12a∶crRNA at the final concentrations of 200 nmol/L and 1 000 nmol/L.The final RT-RAA amplification reaction and CRISPR chromogenic reaction each only took 15 min,and the positive signal could be directly observed under portable blue light irradiation equipment.This method could specifically detect PMMoV,and the detection limit of RNA of pepper samples carrying PMMoV could reach 1.34 pg/μL,which was 1 000 and 10 times more sensitive than that of conventional RT-PCR and real-time RT-PCR.The detection results of 30 samples showed that the RT-RAA-CRISPR/Cas12a assay established in this study could detect PMMoV in leaves,fruits and soils of infected pepper and tomato plants.The established detection system could be used for rapid and sensitive visual detection of pepper mild mottle virus.
万方数据
维普
