Establishment Of Real-time PCR Detection For Toxoplasmosis And A Genotyping Method Of Toxoplasma Gondii Based On HRM

Toxoplasmosis, caused by protozoan parasite Toxoplasma gondii which can infect almost any warm blooded animals, is a global parasitic zoonosis. It is reported that the toxoplasma infection rate of pigs is between 12% and 100% and the fatality rate of infection pigs is more than 60%. This disease may cause severe damages to domestic pig industry. So a fast and precise detection of T. gondii is in urgent need. T gondii has three main distinct lineages(type Ⅰ,Ⅱ and Ⅲ), one of which(type Ⅰ) is highly virulent in mice while the other two attenuated. In this study, We developed real-time PCR detection and constructed a genotyping method by HRM for T.gondii based on the target gene of dense granule protein7(GRA7), using standard strain RH of type Ⅰ, PRU of type Ⅱ and VEG of type Ⅲ.This research established foundation on molecular biological detection, population biology and Epidemiological investigation of T.gondii.Firstly, species-specific primers are designed in conserved regions of GRA7 sequence and Real-time PCR method for Toxoplasmosis is established then applied to clinical diagnosis. The results revealed the specificity that the method cannot amplify Isospora suis, Eperythrozoon suis, Neospora caninum, Ancylostoma caninum or Giardia lamblia from cats except T Gondii.At the same time, this method has high sensitivity that can detect target gene in plasmid sample with the minimal copies of 1 and clinical sample with 10 tachyzoites. Stability is also obvious because the coefficient of variation(CV) of Ct values within the group and between groups were less than 2%. Moreover, it only takes about 100 minutes to accomplish the complete process, from DNA extraction to obtaining final real-time PCR results.360 clinical samples from nine large-scale pig farms around Guangdong province were detected by this new detection method and the results show that Toxoplasma gondii infection rate in the nine farmsis high, with a positive rate at 63%. It is urgent for farm owner to strengthen their supervision and control the disease through medication.Secondly, GRA7 is selected as the target gene in the genotyping method by HRM for three main distinct lineages of T.gondii(type Ⅰ RH strain, type Ⅱ PRU strain and type Ⅲ veg strain). The results showed that the primers HRM-F and HRM-R can clearly distinguish the three genotypes under the optimized reaction conditions. This new HRM genotyping method is stable for good reproducibility of both intra- and inter-assay, and sensitive for its minimum detectable concentration at 5*10-4ng/μL. Nine positive samples from different hosts were detected by this method and four samples of human origin and one of fish were characterized type Ⅱ with Degree of confidence more than 90.82% while two of monkey origin were categorized typeⅠ with more than 91.34%. Genotype of the other two samples from pig were failed to determine, as parasite in the two pig samples might be endemic.