Establishment Of Real-time Fluorescent Quantitative PCR Method For Detecting And Preliminary Application Brucellosis Of Deer

Deer farming industry as the characteristic cultivation has brought huge economic incomefor China’s breeding industry. At present, deer farming in many areas has become an importantindustry for increasing herdsmen’s income, laying economic prosperity in rural, it’s served askey point for strategic adjustment of agricultural structure in some areas of northeast China. Butso far, the disease prevention and control problem of deer is particularly prominent in ourcountry, which seriously restricting the further rapid development of the deer breeding industry.Brucellosis is one of the major infectious diseases of deer, but also a zoonose which hazard tohuman health, it is widely prevalent among our livestock.Deer brucellosis is caused by bovine, sheep and swine type brucella mostly, the otherinfectious types are rare. This study designed general, swine, bovine, sheep type primers andprobes according to the part of highly conserved fragment of the specific genes Omp25c,BruAb2-0168, BMEII0466and BR0952in brucella for detecting all deer brucellosis anddistinguishing their isotype, optimized the reaction system, and screened out the optimalconcentration ratio of primers and probes. The amplification products were cloned into PGEM-Tand pUC57vector to prepare standards and standard curves, then a real-time PCR detectionmethod was established for deer brucella and its sub-type, and its specificity, stability andsensitivity were evaluated. The minimum detectable concentration of this method could beachieved separately to36Copies/μL,12.6Copies/μL,8.7Copies/μL,8.2Copies/μLaccording to the sensitivity of the standard curves, which much higher sensitivity thanconventional PCR.The serum of100parts deer antler blood samples and60parts deer body blood sampleswhich were collected from4deer farms around Changchun city and Liaoyuan city for seraserological detection with rose bengal plate agglutination test (RBPT), and the blood genomesof the samples which test results were positive and suspected positive were extracted forconventional PCR detection and real-time PCR testing. The detected positive samples weremade genotyping identification at the same time. The results showed that35positive samplesand6suspected positive samples were detected by rose bengal plate agglutination test, theknown35positive samples and4positive samples from6suspected positive samples weredetected by conventional PCR, all samples were positive by real-time PCR testing, whichindicated that the real-time PCR technology had strong specificity and high accuracy. Thus, thereal-time PCR detection method developed in this study could not only detect the deer brucellosis but also make genotyping identification, which laid good foundation for censusmonitoring of deer brucellosis and quarantine inspection of deer byproducts.