Establishment Of Two-temperature PCR Method And Real-time Fluorescence Pcr Method For Diagnosing Babesia Caballi And Its Preliminary Application

Babesia caballi is a protozoal disease which is transmit by the ticks and serious influence the health of equine,for its pathogeny is Babesia caballi, the disease will be columned animal disease list by the office international des epizooties,which is a statutory reporting epidemics in our country is classified as Class B animal diseases. the disease is global incidence, characterised by fever, anaemia, icterus and hemoglobinuria, acute cases of death, sub-acute and chronic cases to become the parasite carrier lifelong,this situation cause the local horse and the introduction of the horse cross-infected and recurrent the disease,and causing heavily losses to the horse breeding industry. There is no effective treatment for the disease drugs and vaccines,therefore,the development of new detection technology is the effective way to prevent,control, timely detection,monitoring and integrated control, conducive to the healthy development of the horse industry.(Ⅰ)To establish a rapid,specific and sensitive two-temperature PCR assay for detection of B.caballi,specific primers were designed according to Bc48 gene of B.caballii published in Gen Bank,PCR detection method of B.caballi was established.A gene fragment of 155 bp was amplified by this method,and there was no cross reaction with B.bigemina,T.annulata, T.equi, T.sergenti, and the lowest detection limits of babesia caballi DNA was 5.17×102 copies/μL. Eighty-two blood samples collected from horse in Hejing were detected by the established method.The results showed that the positive rate was69.51%(57/82),while that of blood smear test was 37.8%(31/82).This study indicated that established two-temperature PCR method could be used for early diagnosis of B.caballi with its high specificity and sensitivity.(Ⅱ)A pair of primers and a MGB probe was designed according to the conserved sequence of the Bc48 gene and a MGB FQ-PCR method was establishded for detection of B.caballi.The specificity,sensitivity and reproducibility of the test were conducted.The clinical samples were detected and compared with FQ-PCR and conventional PCR method.The sensitivity of the method was5.17×101copies/μL which was 100 times more than conventional PCR; The FQ-PCRassay sa well as a quantitative standard carve with goood linearity(R2=0.9996) were successfully established.The reproducibility tests in inter-assay and intra-assay indicated that the coefficients of variation were 0.7%and0.48%,respectively,less than 1%;The assay could specifically detected B.caballi and had nagative results for detection Theileria equi、Babesia bigemina、Theilerdia annulata、Theilerdia.sergenti.A total of 90 clinical samples were tested by the established MGB-FQ PCR and the conventional PCR,respectively,the positive rates by the MGB-FQ PCR was 53%,but 30% positive by the conventional PCR.The MGB-FQ PCR assay established in this study was sensitive,specific and stable,and it could be used for B.caballi diagnosis in clinical samples and epidemiological investigation,which will provided a strong guarantee for the tick-brone equine piroplasmosis control.