Rapid and Ultrasensitive Detection of Plasmodium spp. Parasites via the RPA-CRISPR/Cas12a Platform

Microscopic examination of thick and thin blood smearsstainedwith Giemsa dye is considered the primary diagnostic tool for theconfirmation and management of suspected clinical malaria. However,detecting gametocytes is relatively insensitive, particularly in asymptomaticindividuals with low-density Plasmodium infections.To complement existing diagnostic methods, a rapid and ultrasensitivepoint-of-care testing (POCT) platform for malaria detection is urgentlyneeded and necessary. A platform based on recombinase polymerase amplification(RPA) followed by CRISPR/Cas12a (referred to as RPA-CRISPR/Cas12a)was developed and optimized for the determination of Plasmodium spp. parasites, particularly Plasmodium falciparum, using a fluorescence-based assay (FBDA), lateral flowtest strips (LFTS), or naked eye observation (NEO). Then, the establishedplatform was assessed with clinical malaria isolates. Under optimalconditions, the detection threshold was 1 copy/& mu;L for the plasmid,and the limit of detection was 3.11-7.27 parasites/& mu;Lfor dried blood spots. There was no cross-reactivity against blood-bornepathogens. For the accuracies of RPA-CRISPR/Cas12a, Plasmodium spp. and P. falciparum testing were 98.68 and 94.74%, respectively. The method was consistentwith nested PCR results and superior to the qPCR results. RPA-CRISPR/Cas12ais a rapid, ultrasensitive, and reliable platform for malaria diagnosis.The platform requires no or minimal instrumentation for nucleic acidamplification reactions and can be read with the naked eye. Comparedwith similar diagnostic methods, this platform improves the reactionspeed while reducing detection requirements. Therefore, this platformhas the potential to become a true POCT for malaria parasites.