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.
作者
崔艳艳
王晓星
张艳
史柯
菅复春
张龙现
王荣军
宁长申
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)
出处
《中国兽医学报》
CAS
CSCD
北大核心
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
作者简介
崔艳艳（1987-），女，博士研究生。;通讯作者，E-mail：nnl1986@163．com
