啮齿柠檬酸杆菌SYBR Green Ⅰ荧光定量PCR检测方法的建立  

Development of SYBR Green Ⅰ based real-time PCR method for detection of Citrobacter rodentium

作　　者：冯洁[1,2] 谢建云 张泉[1] 魏晓锋 胡建华 高诚 

机构地区：[1]扬州大学兽医学院,江苏扬州225009 [2]上海实验动物研究中心上海市实验动物质量监督检验站,上海201203

出　　处：《中国预防兽医学报》2017年第5期393-397,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：上海市科技创新行动计划(14140900600);上海市科委研发平台专项(15DZ2292400)

摘　　要：为建立快速检测啮齿柠檬酸杆菌(C.rodentium)esp B基因的方法,本研究根据Gen Bank中登录的C.rodentium esp B基因序列设计1对特异性引物,建立了C.rodentium SYBR Green Ⅰ荧光定量PCR检测方法。结果显示,在102拷贝/μL^108拷贝/μL浓度范围内标准曲线循环阈值与模板浓度呈良好的线性关系。该方法仅对C.rodentium进行特异性扩增,而对牛棒状杆菌、鼠伤寒沙门菌、嗜肺巴斯德杆菌、支气管鲍特杆菌、金黄色葡萄球菌、绿脓杆菌、肺炎克雷伯杆菌扩增结果均为阴性,具有良好的特异性。其检测灵敏度可达24拷贝/μL。组内和组间试验变异系数均小于1%,重复性良好。临床样品初步应用结果显示,荧光定量PCR阳性检出率为56.36%(31/55),常规PCR阳性检出率为30.91%(17/55),二者符合率为74.55%。本研究建立的C.rodentium SYBR Green Ⅰ荧光定量PCR检测方法为实验动物C.rodentium的检测和流行病学调查提供了技术支撑。To establish a rapid and specific method for Citrobacterrodentium rodentium detection, a SYBR Green I based real-time PCR method was developed with a pair of primers designed according to the espB gene of C.rodentium. Then a serial dilutions of genomic DNA of C.rodentium were used as standard templates for the real-time PCR to establish the standards curve. The CTs of standard curve had linear relationship with the template concentration. The assay had was specific and reproductive for C.rodentium. No amplification was found from other pathogens, such as Corynebactenum kutscheri, Salmonella typhimunum, Pasteurella pneumotropica, Bordetellabronchisepfica, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiellapneumonia. Sensitivity analysis showed that the detection limit was 24 copies/μL. Results for the clinical samples detection showed that 31 positive samples were detected by the real-time PCR, but only 17 positive samples were detected by normal PCR. The coincidence rate was 74.55%. The results suggested that the SYBR Green I real-time PCR for detection of C.rodentium we established in present study would provide technical support for diagnosis and epidemiological investigation for C.rodenfium.

关 键 词：啮齿柠檬酸杆菌 SYBR Green I 荧光定量PCR 

分 类 号：S852.61[农业科学—基础兽医学]