A novel cross-priming amplification technique combined with lateral flow strips for rapid and visual detection of zoonotic Toxoplasma gondii

Toxoplasma gondii, an obligate intracellular protozoan, infects almost all warm-blooded animals and humans, with felines serving as its sole definitive hosts. Cats release T. gondii oocysts into the environment through feces, contributing to environmental contamination that can lead to toxoplasmosis in humans upon exposure through ingestion of contaminated food, water, or soil. Effective detection of T. gondii in environmental samples is essential for protecting public health and preventing disease transmission. In the present study, we developed a cross-priming amplification (CPA) assay coupled with lateral flow immunoassay strips for the rapid and visual detection of T. gondii in environmental samples. CPA offers simplicity and eliminates the need for complex laboratory equipment. The assay demonstrated high specificity, accurately identifying nine genotypes of T. gondii without cross-reacting with 11 related parasites. Sensitivity testing revealed a detection limit of 1 × 10² copies/μL at the molecular level (plasmid) and 10 oocysts in real-world environmental samples. Furthermore, CPA effectively detected T. gondii in diverse environmental samples, including soil, water, and cat feces, with results consistent with known infection rates. These findings underscore CPA's potential as a reliable, rapid, and accessible tool for detecting T. gondii in environmental settings, contributing to improved public health surveillance and disease prevention.