RPA/CRISPR/Cas12a-Based On-Site and Rapid Nucleic Acid Detection of Toxoplasma gondii in the Environment br

Toxoplasma gondiiis an opportunistic pathogen widely distributed within the world, poses a huge threat to humanhealth, and causes significant economic losses to the livestock industry. Herein, we developed a portable one-pot detection ofT. gondiiby combining recombinase polymerase amplification (RPA) and a clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a system. A glass microfiberfilter device used for thefirst step can efficiently extractT. gondiifrom low-concentration samples. The lyophilized RPA reagents and Cas12a/crRNA reagents are prestored in one Eppendorf tube, and bothreactions can be performed on a low-cost thermal controller (similar to 37 degrees C), avoiding the drawbacks of the step-by-step addition ofcomponents. The developed RPA/CRISPR/Cas12a system exhibits a high selectivity toward the B1 gene amplicon ofT. gondiioverother parasites with a limit of detection of 3.3 copies/mu L. The visual signal readout can be easily realized by afluorometer or lateral-flow strip. A portable suitcase containing the minimum equipment and lyophilized reagents was adopted for the rapid determinationofT. gondiiin heavily polluted landfill leachate. This system presents rapidness, robustness and on-site features for the detection ofnucleic acids of the parasite, making it a promising tool for field applications in remote areas