A novel rapid visual detection assay for Toxoplasma gondii combining recombinase-aided amplification and lateral flow dipstick coupled with CRISPR-Cas13a fluorescence (RAA-Cas13a-LFD)

Abstract

Toxoplasmosis, a parasitic disease resulting from Toxoplasma gondii infection, remains prevalent worldwide, and causes great harm to immunodepressed patients, pregnant women and newborns. Although various molecular approaches to detect T. gondii infection are available, they are either costly or technically complex. This study aimed at developing a rapid visual detection assay using recombinase-aided amplification (RAA) and lateral flow dipstick (LFD) coupled with CRISPR-Cas13a fluorescence (RAA-Cas13a-LFD) to detect T. gondii. The RAA-Cas13a-LFD assay was performed in an incubator block at 37 °C within 2 h, and the amplification results were visualized and determined through LFD by the naked eye. The detection limit was 1 × 10−6 ng/μL by our developed RAA-Cas13a-LFD protocol, 100-fold higher than that by qPCR assay (1 × 10−8 ng/μL). No cross-reaction occurred either with the DNA of human blood or Ascaris lumbricoides, Digramma interrupta, Entamoeba coli, Fasciola gigantica, Plasmodium vivax, Schistosoma japonicum, Taenia solium, and Trichinella spiralis, and the positive rate by RAA-Cas13a-LFD assay was identical to that by qPCR assay (1.50% vs. 1.50%) in detecting T. gondii infection in the unknown blood samples obtained from clinical settings. Our findings demonstrate that this RAA-Cas13a-LFD assay is not only rapid, sensitive, and specific and allows direct visualization by the naked eye, but also eliminates sophisticated and costly equipment. More importantly, this technique can be applied to on-site surveillance of T. gondii.