嗜吞噬细胞无浆体SYBR GreenⅠ荧光定量PCR检测方法的建立及应用  

Development and application of SYBR GreenⅠ real-time fluorescent quantitative PCR for detection of Anaplasma phagocytophilum

作　　者：崔艳艳[1] 王晓星[1] 张艳[1] 史柯[1] 菅复春[1] 张龙现[1] 王荣军[1] 宁长申[1] CUI Yan-yan;WANG Xiao-xing;ZHANG Yan;SHI Ke;JIAN Fu-chun;ZHANG Long-xian;WANG Rong-jun;NING Chang-shen(College of Animal Science and Veterinary Medicine, Henan Ag ricultural University, Zhengzhou 450002, Chin)

机构地区：[1]河南农业大学牧医工程学院

出　　处：《中国兽医学报》2018年第4期722-726,735,共6页Chinese Journal of Veterinary Science

基　　金：国家现代肉羊产业技术体系基金资助项目(nycytx-39);河南省教育厅现代畜牧业河南省协同创新中心资助项目

摘　　要：为建立荧光定量PCR方法检测嗜吞噬细胞无浆体（Anaplasma phangocytophilum），针对GenBank A．phagocytophilum 16S rRNA基因保守序列（JN558815），设计1对引物1—25F／183—205R。以已知引物EE1／EE2和该引物进行巢式PCR扩增出预期大小片段（205bp），构建合有16srRNA基因的重组质粒，以质粒为模板构建了标准曲线。以1—25F／183—205R为荧光定量PCR引物，建立A．phagocyto philum荧光定量PCR检测方法，并进行特异性、敏感性、重复性试验和临床样本检测。结果表明，该方法在6．4×10^3～6．4×10^8copies／μL相关系数为0．99，显示出良好的线性关系；所建方法能特异地检出A．phagocytophilum，对绵羊无浆体、牛无浆体和环形泰勒虫基因组DNA和灭菌双蒸水均无交叉反应；可检测到6．4×10^2copies／μL的标准质粒DNA，比常规PCR敏感性高100倍；批内及批间变异系数均低于2．0％，具有较高的重复性和稳定性；利用该方法对30份羊血液临床样品检出率比巢式PCR高16．67％。该研究为A．phagocytophilum临床检测和定量分析病原感染程度奠定理论基础。In order to develop SYBR Green I fluorescent quantitative PCR for detection Anaplas- ma phagocytophilum ,one pair of primers was designed based on conserved sequence of 16S rRNA gene in GenBank. A expected size fragments（205 bp） was amplified by nested PCR with the two sets of primer,EE1/EE2 and 1-25 F/183-205R. Constructed a recombinant plasmid containing 16S rRNA gene was as template to set up RT-PCR standard curve. The method of RT-PCR for detec- tion A. phagocytophilum was established based on a pairs of primer F/183-205R 1-25, meanwhile specific assay, sensitivity assay, reproducibility and clinical application of the assay were deter- mined. The results indicated that standard curve showed a good linear relationship with template concentration ranging from 6. 4 × 10^3-6. 4 × 10^8 copies/μL. Then, the specific and sensitive tests showed that the assay was only detected A. phagocytophilum with the detection limit of 6.4 × 10^2 copies/μL,but had no cross-reaction with A. ovis,A, boris, T. annulata and sterilization ddH20. The variations of both inter-and intra-assay were less than 2.0 %. The detection rate of this meth- od is high than convention PCR assay（16. 67%） for detecting 30 clinical samples. This study developed the basis for detection of A. phagocytophilum and analysis of the degree of pathogen infection.

关 键 词：嗜吞噬细胞无浆体 荧光定量PCR 16S RRNA基因 

分 类 号：S852.62[农业科学—基础兽医学] R535[农业科学—兽医学]