Establishment And Application Of Direct PCR Method For The Detection Of T.oriental

Theileria Oriental is an intracellular protozoan parasite transmitted by ticks,which is widely distributed all over the world.The pathogen can cause bovine Theileria Orientellosis,and the main clinical symptoms are fever,hemolytic anemia,decreased productivity,miscarriage or death of dairy and beef cattle,causing economic loss and harm to cattle industry.At present,blood direct PCR detection method has been developed and applied in animal disease epidemiology research because it does not need to extract DNA and directly use blood to save time and reduce cost.Therefore,the direct PCR detection method was established in this experiment,which was based on the blood of cattle infected with Theileria Orientellosis.In this paper,we first designed a pair of specific primers according to the p33 gene sequence[DQ078264.1]of Theileria oriental published on Genbank,and amplified a band of the size of 416 bp by blood direct PCR reaction system with blood as template.The homology between the nucleotide sequence obtained by sequencing and the p33 gene published in the gene library was 99%,and the target fragment was confirmed to be the p33 gene.In addition,the primers designed in this experiment failed to amplify the target bands of Toxoplasma gondii,Neosporidium caninum and Bovine Mycoplasma suis,indicating that the method has good specificity.At the same time,the maximum dilution ratio of 1 ?L blood can be detected by this method is 40 times.It shows that the method has good sensitivity.In addition,5 samples of Theileria Oriental positive blood samples were tested by direct PCR and routine PCR,respectively.The results of three repeated tests showed that the method had good repeatability.A total of 210 suspected bovine anticoagulant blood samples from several counties and cities in Jilin Province were collected for direct PCR detection.Compared with the conventional PCR method,the advantages of this method in diagnosis and detection were determined.The results showed that the total positive rate of direct PCR was 30.0%,the total positive rate of routine PCR was 25.7%,the coincidence rate of the two methods was 94.0%,and the Kappa value of concordance analysis was 0.85.The positive and negative coincidence rates were 80.0%and 92,0%respectively.The positive rate in Tonghua area was the highest(75.0%),while that in Taonan area was the lowest(12.0%).There was statistical difference in the positive rate among different areas(P<0.05).The results were consistent with the results reported by Hu Shiyue,et al.The infection rate was 21.1%?75.9%in Jilin Province.The results show that the method is worth popularizing and applying.Therefore,a simple,quickly,sensitive and specific rapid detection method can be used for the diagnosis and epidemiological investigation of Theileria Orientellosis.