Designing and developing of high-resolution melting technique for separating different types of Toxoplasma gondii by analysis of B1 and ROP8 gene regions

Abstract

BackgroundDetermination of Toxoplasma gondii genotypes plays an important role in the health management and epidemiology of toxoplasmosis. We developed HRM analysis to differentiate genotypes of T. gondii using the B1 and ROP8 genes, through comparing the sensitivity and specificity of both genes and methods used for the detection of T. gondii. MethodsA total of 96 DNA samples of muscle tissue of livestock and poultry brain tissue with three standard strains RH (type I), PRU (type II) and VEG (type III) were prepared and analyzed. Three methods of nested PCR, PCR-PCR and nested-qPCR-HRM were used. Specific new primers were designed and synthesized for developing HRM. Thirty positive samples obtained from nested-qPCR-HRM were sequenced (18 B1 and 12 ROP8). ResultsOverall, 87 infected samples were identified using both genes. Through the B1 gene, we could separate type I (Tm = 84.8 °C) from II/III types (Tm = 84.6 °C). Also, the ROP8 gene could separate type II (Tm = 84.5 °C) from I/III types (Tm = 84.12 °C). Highest sensitivity (100%) and specificity (78.72%) were observed by nested-qPCR-HRM assays of the B1 and ROP8 genes than by other methods, respectively. Thus, the B1 gene can be used to most accurately detect T. gondii, while the ROP8 gene was more appropriate for T. gondii genotyping. PCR-sequencing results were consistent with HRM results in most selected samples. ConclusionHRM analysis is a powerful diagnostic tool for rapid detection and determination of main clonal lineages, and even unusual T. gondii genotypes.