Development And Primary Application Of PCR-Elisa Method For The Detection Of P33 Gene T.Sergenti

Theileriosis is parasitic on cattle red blood cells, macrophages and lymphocytes, belonging to Theileriidae, Theileria genus Theileria blood worms protozoosis, medium for longicornis.High relapsing fever, anemia, weight loss, bleeding, and surface lymph nodes and other symptoms are exhibited clinically infected with this parasite in cattle.The disease can affect milk out of postpartum cows and reduces the survival rate of born calves,very serious harm, mainly in the Northeast Asia region, bringing serious economic losses to the cattle industry.In recent years, the rapid development of the global economy, the cattle industry has also evolved,at the same time more theileriosis been reported, has been brought serious economic losses to the regions and the country’s livestock,has been widespread concern in the veterinary sector.Therefore, it is imperative to monitor, control and monitoring theileriosis reduce its incidence.To this end, the present study established a simple, specific and sensitive theileriosis PCR-ELISA detection method for epidemiological survey and provide diagnostic tools.First, we designed a pair of conventional primers based on Theileria gene sequence[DQ078264.1]published on Genbank,Biotin and digoxin are labeled in conventional primers downstream of the 5’, a pair of primers were labeled with non-radioactive substance by synthesized. Genomic DNA was extracted from Theileriosis positive anticoagulant after blood smears,as a templates combination was designed primers designde primers get on conventional PCR, to obtain the disease object gene fragment of the p33,after purification recovery,cloned into the vector of pMD-19T simple,Plasmids were identified by PCR and double endonuclease digestion appraisal,determining a positive result, sent to the biological company sequencing,etablish PCR detection method of specific.According to the basic steps of.PCR-ELISA method,to optimize the streptavidin-biotin concentration, closing time, dilution of the PCR product, dilution of antibodies and color development time,optimum conditions of the PCR-ELISA reaction.Forty parts of theileriosis negative samples were determined to be detected by PCR, measuring OD450nm value, critical values calculated as criteria in accordance with the formula x+3SD.Dilution theileriosis genomic DNA, the sensitivity of this method was measured under optimal reaction conditions.As a template of Toxoplasma gondii, Neospora, Babesia and other genomic DNA to detect the specificity of the method under the optimum reaction conditions.To detect the stability of such a method by the same time coated microtiter plates repeatability and at different times coated microtiter plates reproducibility of experiments.Take 112 cattle anticoagulant to be tested to detect compared with conventional PCR methods to determine the application of this method in the clinic. The results showed that the stripe size is about 421 bp after routine PCR detection, Homology of 99% compared with the gene sequence of sergenti Theileria published on Genbank.The amplified fragment was theileriosis.Optimize all aspects of the experiment,obtained was diluted to 5 ug/mL streptavidin-biotin,PCR products were diluted 30-fold,2000-fold diluted of HRP-labeled digoxin antibody,60 min closed time,chromogenic substrate at 15 min, for the optimal reaction conditions.Obtain the critical value of 0.174 based on the 40 parts identified as theileriosis negative samples by PCR.when the sample OD450nm value is greater than 0.174, theileriosis positive, otherwise it is negative.Theileriosis genomic DNA owest concentration is 18 fg/μL by PCR-ELISA method detected,Sensitivity 10 times higher than the conventional PCR method detected 0.2 pg/μL,specific stronger,no cross-reactivity between cow Toxoplasma gondii, Neospora, Babesia, etc.Positive rate of 34.8%,28.6% higher than conventional PCR methods.After conducting clinical testing on 112 parts by bovine serum to be tested.The experiment to establish a specific, sensitive, safe, time-saving method of diagnosis of theileriosis, and provides an easy way to theileriosis diagnosis and epidemiological investigation.