The Establishment Of Multiple PCR Detection Method For Cattle Neospora Caninum And Theileria Sergenti

Theileriosis is one of blood protozoal disease, caused by Theileriafamify, Theileria genus, Theileria sergenti. Cattle Neosporosis is akind of protozoal disease that is caused by Neospora caninum orNeosporalike that parasitized in cattle body.This research takes use of the DNASIS software to analyze thedimeride, the homology and complementarity of the primer, which aredesigned by p33 surface protein gene order of the Theileria sergenti andspecies-specificity gene fragment Nc-5 sequence that was from GenBank,in order to avoid the formation of the stable primer dimer and higherhomology or complementarity among the primers. At the same time, basedon the rule PCR of the established cattle Theileria sergenti and cattleNeospora caninum, optimizing the concentration of primer, MgCl₂, dNTPand the number of circulatory times and so on, at last we establish themultiple PCR in order to detcet cattle Theileria sergenti and cattleNeospora caninum.The system specific amplification fragments are Theileria sergenti864bp and Neospora caninum 338bp respective. The results of systemoptimizing: the best primer concentration are Theileria sergenti0.2pmol/mL, Neospora caninum 0.2ug/mL; the best concentration of dNTPis 1.6mmol/L; the best reaction pattern of multiple PCR is 94℃5min,94℃30s, 56℃40s, 72℃1min, in all there are 30 circulations, then72℃10min for extention, at last 4℃7min for ending the PCR amplification;Taking use of the optimizing PCR amplification system, we get the 864bp and 338bp specific electrophoresis band in accord with the design ofexperiment. The results of sensitivity experiments showed that multiplePCR could detect the nucleic acid template of concentration of Theileriasergenti was 160fg/mL, Neospora caninum was 18pg/mL; taking use of DNAof anticoagulated blood that infected Eperythrozoon suis,cattleNeospora caninum,the anticoagulated blood that infected cattleTheileria sergenti,T.gondii,cattle Clostridieum welchii,cattleBacillus tuberculosis,cattle Baoterium coli to have PCR amplification,in order to determine the specificity of multiple PCR, the results showedthat Neospora caninum and Theileria sergenti all amplificated the samenucleonic acid fragments in accord with the design of experiments; butthe contrast pathogen did not amplificate any nucleic acid fragments;Taking use of the cattle disease material that was Neospora caninumpositive by rule PCR detection and cattle anticoagulated blood that wasTheileria sergenti positive by rule PCR detection, the results showedthat the coincidence was 100% between the multiple PCR detection resultsand rule PCR detection results. All above results indicated that themultiple PCR amplification method this research established has specific,sensitive and Volant grace, it is suitable to diagnose and infectedmonitoring the cattle Theileriosis and cattle Neosporosis.