RPA-CRISPR/Cas12a-LFA combined with a digital visualization instrument to detect in stray dogs and cats in Zhejiang province, China.

UNLABELLED

, which causes toxoplasmosis, is prevalent in warm-blooded animals, such as cats, dogs, and humans. causes economic losses to livestock production and represents a potential risk to public health. Dogs and cats are common hosts in the epidemiology of toxoplasmosis. The current molecular diagnostic tools for infection require high technical skills, a laboratory environment, and complex instruments. Herein, we developed a recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) assay to detect . The lowest limit of detection of the assay was 31 copies/μL for the gene. In addition, we established a visual RPA-CRISPR/Cas12a lateral flow band assay (RPA-CRISPR/Cas12a-LFA) combined with a digital visualization instrument, which minimized the problem of false-negative results for weakly positive samples and avoided misinterpretation of the results by the naked eye, making the LFA assay results more accurate. The assay established in this study could identify within 55 min with high accuracy and sensitivity, without cross-reaction with other tested parasites. The developed assay was validated by establishing a mouse model of toxoplasmosis. Finally, the developed assay was used to investigate the prevalence of in stray cats and dogs in Zhejiang province, Eastern China. The positive rates of infection in stray cats and dogs were 8.0% and 4.0%, respectively. In conclusion, the RPA-CRISPR/Cas12a-LFA is rapid, sensitive, and accurate for the early diagnosis of , showing promise for on-site surveillance.

IMPORTANCE

is a virulent pathogen that puts millions of infected people at risk of chronic disease reactivation. Hosts of are distributed worldwide, and cats and dogs are common hosts of . Therefore, rapid diagnosis of early infection and investigation of its prevalence in stray dogs and cats are essential. Here, we established a visual recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a-assay combined with a lateral flow band assay and a digital visualization instrument. Detailed analyses found that the assay could be used for the early diagnosis of without false-negative results. Moreover, we detected the prevalence of in stray cats and dogs in Zhejiang province, China. Our developed assay provides technical support for the early diagnosis of and could be applied in prevalence surveys of in stray dogs and cats.