[Establishment and preliminary evaluation of a fluorescent recombinase-aided amplification/CRISPR-Cas12a system for rapid detection of ].

Objective: To establish a fluorescent assay for rapid detection of based on recombinaseaided amplification (RAA) and CRISPR-Cas12a system,and to preliminarily evaluate the diagnostic efficiency of this system.

Methods: The ribosomal RNA () gene of was selected as the target sequence, and three pairs of RAA primers and CRISPR-derived RNA (crRNA) were designed and synthesized. The optimal combination of RAA primers and crRNA was screened and the reaction conditions of the system were optimized to create a fluorescent RAA/CRISPR-Cas12a system. The plasmid containing gene of the strain 3D7 was generated, and diluted into concentrations of 1 000, 100, 10, 1 copy/μL for the fluorescent RAA/CRISPR-Cas12a assay, and its sensitivity was evaluated. The genomic DNA from , , , hepatitis B virus, human immunodeficiency virus and was employed as templates for the fluorescent RAA/CRISPR-Cas12a assay, and its specificity was evaluated. Fifty malaria clinical samples were subjected to the fluorescent RAA/CRISPR-Cas12a assay and nested PCR assay, and the consistency between two assays was compared. In addition, strain 3D7 was cultured . Then, the culture was diluted into blood samples with parasite densities of 1 000, 500, 200, 50, 10 parasites/μL with healthy volunteers' O-positive red blood cells for the RAA/CRISPR-Cas12a assay, and the detection efficiency was tested.

Results: The Pf-F3/Pf-R3/crRNA2 combination, 2.5 μL as the addition amount of B buffer, 40 min as the RAA reaction time, 37 °C as the reaction temperature of the CRISPR-Cas12a system were employed to establish the fluorescent RAA/CRISPR-Cas12a system. Such a system was effective to detect the plasmid containing gene of the strain 3D7 at a concentration of 1 copy/μL, and presented fluorescent signals for detection of , but failed to detect , , , , hepatitis B virus or human immunodeficiency virus. The fluorescent RAA/CRISPR-Cas12a system and nested PCR assay showed completely consistent results for detection of 50 malaria clinical samples ( = 1.0, < 0.001). Following 6-day culture of the strain 3D7, 10 mL cultures were generated and the fluorescent RAA/CRISPR-Cas12a system showed the minimal detection limit of 50 parasites/μL.

Conclusions: The fluorescent RAA/CRISPR-Cas12a system is rapid, sensitive and specific for detection of , which shows promising value for rapid detection and risk monitoring of .