Development and evaluation of an antisense PCR assay for Toxoplasma gondii detection in domestic cats

As definitive hosts for Toxoplasma gondii, cats serve as critical reservoirs for human toxoplasmosis transmission. Therefore, rapid and accurate diagnosis of cat T. gondii infection is essential for public health. In this study, we developed an antisense PCR assay targeting the B1 gene of the T. gondii. Antisense primers were used to effectively suppress residual outer primer activity during the second-stage amplification process in a single-tube nested PCR format. Sensitivity analysis demonstrated a detection limit of 0.1 pg/μL T. gondii DNA, representing a 10-fold improvement over conventional PCR (1 pg/μL). Specificity testing confirmed no cross-reactivity with other cat pathogens. Experimental validation using 33 experimentally infected cat blood samples revealed 24 positive cases by antisense PCR (72.73 %, 24/33), significantly outperforming conventional PCR (36.36 %, 12/33; Kappa = 0.353). McNemar's test showed a statistically significant difference (P < 0.001). This antisense PCR method provides a sensitive, contamination-resistant, and cost-effective tool for diagnosing cat toxoplasmosis, with potential applications in clinical laboratories.