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

Abstract

Toxoplasma gondii is an opportunistic pathogen widely distributed within the world, poses a huge threat to human health, and causes significant economic losses to the livestock industry. Herein, we developed a portable one-pot detection of T. gondii by combining recombinase polymerase amplification (RPA) and a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. A glass microfiber filter device used for the first step can efficiently extract T. gondii from lowconcentration samples. The lyophilized RPA reagents and Cas12a/crRNA reagents are prestored in one Eppendorf tube, and both reactions can be performed on a low-cost thermal controller (∼37 °C), avoiding the drawbacks of the step-by-step addition of components. The developed RPA/CRISPR/Cas12a system exhibits a high selectivity toward the B1 gene amplicon of T. gondii over other parasites with a limit of detection of 3.3 copies/μL. The visual signal readout can be easily realized by a fluorometer or lateralflow strip. A portable suitcase containing the minimum equipment and lyophilized reagents was adopted for the rapid determination of T. gondii in heavily polluted landfill leachate. This system presents rapidness, robustness and on-site features for the detection of nucleic acids of the parasite, making it a promising tool for field applications in remote areas.

Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and applications

“…established a portable one-pot assay for Toxoplasma gondii using RPA-CRISPR/Cas2a with a lower limit of detection of 3.3Copes/μl. A portable suitcase was also designed to meet the needs of on-site detection ( Lei et al., 2022b ). RPA had been successfully applied to detect many parasites, but the application was not yet widespread.…”

Research progress of CRISPR-based biosensors and bioassays for molecular diagnosis

“…Driving a pair with different sgRNAs of Cas9 generates dsDNA with sticky ends, ligated to a hairpin-structure substrate, which subsequently generates a large number of short fragments that report the electrical signal of the detected DNA (Dai et al, 2020). Huang's team developed a dCas9-ECL probe for sensitive and single-base specific DNA detection with good resistance to nonspecific interference and a low false positive detection rate (Wu L. et al, 2022). This shows that electrochemical biosensors can modularize the circuitry with better programmability.…”

“…Nevertheless, current research on non-nucleic acid targets is also of considerable interest. Many detection systems have developed, especially in the food and environmental fields, to detect pathogenic small molecules or parasites (Lei et al, 2022;You et al, 2022), or pathogens in agriculture (Jiao et al, 2022;Zhu et al, 2022).…”

Development of CRISPR-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry

“…Toxoplasma gondii is a globally distributed protozoan parasite, causing life-threatening consequences in immunocompromised patients [ 140 ] as well as abortions and stillbirths in livestock [ 141 , 142 ]. In combination with RPA or RAA, CRISPR-Cas12a/Cas13a based methods have been reported for quick in-field detection of T. gondii [ 91 , 92 , 93 ]. The authors demonstrated that the RAA-Cas12a/Cas13a systems have advantages over conventional PCR-based method in terms of conveniency and sensitivity [ 92 , 93 ].…”

“…The authors demonstrated that the RAA-Cas12a/Cas13a systems have advantages over conventional PCR-based method in terms of conveniency and sensitivity [ 92 , 93 ]. In terms of reducing cross-contamination and cost, the RPA-CRISPR/Cas12a assay (single-tube strategy and 100 nM of Cas12a) is more advanced than the RAA-CRISPR/Cas12a assay (two separate processes and 800 nM of Cas12a) [ 91 ]. Applications of these novel methods potentially contribute to the control of toxoplasmosis in humans and animals.…”