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

作　　者：张志君[1] 李兆才[1] 娄忠子[1] 周继章[1] ZHANG Zhi-jun;LI Zhao-cai;LOU Zhong-zi;ZHOU Ji-zhang(State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]中国农业科学院兰州兽医研究所,兰州730046

出　　处：《中国人兽共患病学报》2018年第3期224-229,共6页Chinese Journal of Zoonoses

基　　金："十三五"国家重点研发计划(No.2016YFD0500907);甘肃省科技支撑计划(No.1504NKCA054)联合资助~~

摘　　要：目的建立荧光定量PCR(qPCR)对流产衣原体的检测方法。在流产衣原体灭活疫苗的生产中,代替具有主观判断影响的涂片染色疫苗效价检测方法。方法根据流产衣原体富含半胱氨酸的胞质蛋白(envB)基因保守序列设计特异性引物,利用EnvB-PMD19T阳性质粒为参考标准品,优化了反应条件,进行了敏感性、特异性及重复性试验,检测了感染流产衣原体小鼠的菌体载量。结果以重组质粒为标准品建立的标准曲线在1×10~2 copies/μL^1×10~6 copies/μL内呈现良好的线性关系,扩增效率为97%。灵敏度试验表明,该检测方法的最低检出限低至10copies/μL。组内、组间重复性试验的变异系数均小于2%,具有良好的重复性和稳定性。消化道检测到的菌体载量高于腹腔注射的菌体载量。结论本研究建立的方法能够有效的检测小鼠相应脏器的菌体含量,为流产衣原体灭活疫苗的效检提供可靠的检测方法。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,sensitivity 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 1×10 2 copies/μL to 1×10 6 copies/μL with the correlation coefficient of 97%,sensitive for detecting C.abortus with the detection limit of 10 copies/μL,and repeatable 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 

分 类 号：R374[医药卫生—病原生物学]