High-resolution melting (HRM)-based genotyping of Toxoplasma gondii in meat products: comparative evaluation of...

Consumption of raw or undercooked meat is a major route of transmission of Toxoplasma gondii (T. gondii) to humans worldwide. This study aimed to develop and evaluate high-resolution melting (HRM) assays targeting two novel polymorphic regions of the rhoptry protein 18 (ROP18) gene (106-bp and 125-bp) for genotyping T. gondii in meat products, in comparison with the B1 and ROP5 genes. A total of 64 samples, including 40 commercial meat products (24 sausages, 9 hams, and 7 hamburgers), 24 tissue samples from livestock (cattle, goat, and sheep) and poultry brains, and three reference strains (RH, ME49, and VEG), were collected from Qom, Iran. Following pepsin digestion and DNA extraction, samples were analyzed by nested/semi-nested PCR and HRM. The B1 gene detected T. gondii in 35 samples (87.5%), whereas ROP5 identified 18 (45%). HRM based on B1 and ROP5 partially discriminated genotypes I, II, and III, while the 106-bp region of ROP18 successfully resolved all three canonical types (Tm: I = 86.54 °C, II = 85.42 °C, III = 86.19 °C). The 125-bp region distinguished type III (86.36 °C) from types I/II (84.90 °C). Thirteen ROP5-positive samples were sequenced and submitted to GenBank (MW521195-MW521220), revealing mixed (I/II/III, I/III) and atypical genotypes. Phylogenetic analysis confirmed the ability of ROP5 to cluster type II separately. Taken together, our findings demonstrate that ROP18 gene regions are superior to B1 and ROP5 for precise, cost-effective HRM-based genotyping of T. gondii in complex meat matrices, a methodology increasingly validated in high-impact molecular diagnostic platforms.