中国人兽共患病学报2018，Vol.34Issue(3)：224-229,6.DOI:10.3969/j.issn.1002-2694.2018.00.023
流产衣原体荧光定量PCR方法的建立及小鼠感染菌体载量测定
Establishment of detection method for Chlamydophila abortus by using SYBR green real-time PCR and determination of bacterial load in mice
摘要
Abstract
We established a fluorescent quantitative PCR (qPCR)method for the detection of Chlamydophila abortus (C. abortus),and replaced the method of smear staining which has subjective influence on the detection of C.abortus inactivated vaccine titer.According to the conserved sequence of the large cysteine-rich periplasmic protein(envB)of C.abortus,a specific primer was designed and the EnvB-PMD19T positive plasmid was used as the reference standard,optimization condition,sensi-tivity assay,specificity assay,repeatability assay and the bacteria loads of organs from mouse have been done.The results showed that the standard curve established with positive plasmid had a liner response from 1×102copies/μL to 1×106copies/μL with the correlation coefficient of 97%,sensitive for detecting C.abortus with the detection limit of 10 copies/μL,and re-peatable and stable with the coefficients of variation less than 2%.According to the result,the established method can detect the bacteria loads in organ of mouse,which provide a reliable method for evaluation of inactivated C.abortus vaccine.关键词
Balb/c小鼠/流产衣原体/实时荧光定量PCR/绿色荧光染料Key words
Balb/c mouse/Chlamydophila abortus/real-time PCR/SYBR Green dye分类
医药卫生引用本文复制引用
张志君,李兆才,娄忠子,周继章..流产衣原体荧光定量PCR方法的建立及小鼠感染菌体载量测定[J].中国人兽共患病学报,2018,34(3):224-229,6.基金项目
"十三五"国家重点研发计划(No.2016YFD0500907)和甘肃省科技支撑计划(No.1504NKCA054)联合资助Supported by the State Key Development Program (No.2016YFD0500907)and the Gansu Science and Technology Support Plan (No.1504NKCA054) （No.2016YFD0500907）
