Development of a novel multiplex PCR assay for the specific identification of Toxocara canis, Toxocara cati and Toxascaris leonine in dogs and cats.

Parasitic diseases are among the common health concerns in dogs and cats, with ascaridoid nematodes representing one of the major groups of zoonotic parasites. Three ascaridoids-Toxocara canis, Toxocara cati, and Toxascaris leonina-commonly infect these hosts. Effective diagnostic techniques are important for systematics, epidemiology, and clinical management. In this study, a multiplex PCR (mPCR) assay was developed for the simultaneous and specific identification of three ascaridoids. Specific primers targeting conserved mitochondrial regions were designed. Sensitivity was evaluated using serially diluted standard DNA samples, and specificity was assessed by testing cross-reactivity against a Madin-Darby canine kidney cell line, Ancylostoma caninum, Dipylidium caninum, Trichinella spiralis, and Escherichia coli. The reliability of the assay was validated using 60 clinical samples collected from dogs and cats. The experimental results demonstrated that the detection limit of the assay was 10² copies/µL for a single ascaridoid and 10 ³ copies/µL for the simultaneous detection of three ascaridoids. Furthermore, the assay exhibited good specificity and reliability. The assay provides a practical tool for diagnosing and monitoring major ascaridoid infections in dogs and cats, with potential applications in source tracing of human toxocariasis.