Establishment And Application Of Real-Time PCR Detection Method Of Theileria Sergenti

Theileria sergenti is a tick-borne Apicomplexan protozoa.Clinically,theileriosis causes chronic anemia,fever,icterus and even death.Currently,Theileria sergenti is wide-spread among in cattle all over the world.With the development of trade globalization and cattle industry, the trade activities of cattle and cattle products all over the world, Theileria sergenti have been found in different continents or regions. Therefore, it is urgently considered to develop effective diagnostic method for the monitoring and prevention to Theileria sergenti infection.Real-time fluorescent quantitive PCR is a laboratory technique of molecular biology based on the PCR, which is used to calculate original number of the DNA target and supervise the whole PCR process by adding some fluorescence group in the reaction. Since1996, with the development of the real-time fluorescent quantitive PCR, this technology has been widely used in different fields. The type of real-time fluorescent quantitive PCR technique used depends on the DNA sequence in the samples, the technique can either use non-specific fluorochromes or hybridization probes. SYBR Green PCR method is the dsDNA dyes such as SYBR Green binding to all dsDNA PCR products. However, TaqMan PCR method is fluorescent reporter probes detecting the DNA which containing the probe sequence.In the current research, we designed two pairs of specific primers according to P33gene sequence of Theileria sergenti, and then the standard DNA samples were extracted from cattle blood. The concentration of the corrected standard DNA samples were detected and diluted for the SYBR Green PCR and TaqMan PCR amplification. In the study, the primers concentration is optimized, the sensitivity and the specificity and the repeatability of the two assays were evaluated.The results showed that using SYBR Green PCR method the optimal primers concentration of is0.4μmol/L, the linear range of detection is2.26×108copies/μL2.26×100copies/μL. Using TaqMan PCR method, the optimal concentration of primers and probe is0.4μmol/L and0.3μmol/L, the linear range of detection is2.26×108copies/μL-2.26×100copies/μL. The melting curve analysis showed that SYBR Green PCR and TaqMan PCR method had good specificity. The critical value (CV) of the Intrasession and intersession coefficient of variation and coefficient of stability is less than5%, which showed two PCR methods owing reproducibility.We compared the developed TsPl-SYBR Green PCR and TsP1-TaqMan PCR in the study with common PCR. The results showed that the three methods have good specificity, the two fluorescent quantitative PCR in the study showed higher sensitivity about10,000times than common PCR. We collected79cattle blood samples from Hunchun and Songyuan, and then detected the infection rate of Theileria sergenti. The positive rate of Theileria sergenti is36.71%,46.84%, and46.84%using common PCR, TsP1-SYBR Green PCR, and TsPl-TaqMan PCR, respective.In the study, we developed the SYBR Green PCR and the TaqMan PCR to detect Theileria sergenti. The two methods not only overcomes the disadvantages of other diagnosis method, such as the low sensitivity, cross-contamination, poor specificity, false positive and long detection time, but also increase sensitivity, high-throughput and accurate quantitative detection. The two PCR methods not only provide a well services to monitor and control Theileria sergenti, but also to evaluate the therapeutic effect of drugs and immune effects of vaccines.