牛呼吸道综合症多重PCR检测方法的建立

Establishment of Multiple PCR Detection Method of Bovine Respiratory Disease Complex

为了建立针对BRDC常见的主要病原牛黏膜病病毒(BVDV)、牛副流感病毒3型(BPIV-3)和牛呼吸道合胞体病毒(BRSV)的一步法多重RTPCR,引入了新型的具有反转录酶和DNA聚合酶活性的r Tth DNA聚合酶,通过对退火温度和引物浓度的优化,敏感性和特异性的检测,最终确定最佳退火温度为54.5℃,最佳引物浓度为0.6 u M。对BPIV-3、BRSV和BVDV的最小检测量分别为162、16和21个TCID50/0.1 m L。与牛传染性鼻气管炎病毒、冠状病毒、腺病毒,以及多杀性巴氏杆菌、肺炎链球菌、化脓隐秘杆菌、溶血性曼氏杆菌等没有交叉反应。建立起的m RT-PCR检测方法可用于鼻腔分泌物的检测,为进一步m RT-PCR试剂盒的组装奠定了基础。

Bovine respiratory syncytial virus,bovine viral diarrhoea virus and bovine parainfluenza virus 3 were three of the major viruses associated with BRDC. In this study,one-step multiple RT-PCR （mRT-PCR） was established for detecting these three pathogens. The rTth DNA polymerase, a new type of reverse transcriptase with the activity of DNA polymerase,was used in the study. Through the optimization of annealing temperature and concentration of primers,sensitivity and specificity of detection,finally the optimum annealing temperature was 54.5 ℃ ,the best primer concentration was 0.6 uM. The BPIV-3,BRSV and BVDV were minimum detectable quantity 162,16, and 21 respectively TCID50/0.1 mL.Without crossing reaction to infectious bovine rhinotraeheitis virus, coronavirus, adenovirus, and pasteurella muhocida, streptococcus pneumoniae, arcanobacterium pyogenes, Mannheimia haemolytica,etc. For clinical validation,the established mRT-PCR could be used to the detection of nasal secretions in clinical samples from cattle. The study laid further a solid foundation for the mRT-PCR kit assembly.