摘要
为建立检测贝氏柯克斯体(Coxiella burnetii)实时荧光定量PCR方法,根据GenBank上收录的C.burnetii RSA439株com1基因序列(CP040059.1)保守区设计特异性引物和探针,并构建重组质粒及优化反应条件,以期建立一种检测贝氏柯克斯体的Taq Man荧光定量PCR方法。结果显示,该方法所建立标准曲线线性关系良好;敏感性试验结果显示,最低检出限为2.45×10^(2) copies/μL,与PCR最低检出限为2.45×10^(4) copies/μL相比较,灵敏度提高了100倍;对羊支原体、羊流产衣原体、羊布鲁氏菌等均无交叉反应,批内和批间重复性试验结果稳定,变异系数均小于3%,说明特异性、重复性好;利用该方法对收集的126份样品进行检测,结果显示样品阳性率为9.52%,常规PCR检测阳性率为7.14%,符合率为90.47%。该方法可为贝氏柯克斯体临床上快速检测及防控提供技术支持。
To establish the Taq Man real-time PCR detection method for Coxiella burnetii,we referred to the C.burnetii included in GenBank.We used the conserved region of the RSA439 strain com1 gene sequence(CP040059.1)to design specific primers and probes,construct recombinant plasmids,and optimize reaction conditions in order to establish a Taq Man real-time PCR method for Coxiella burnetii.The results showed that the standard curve established by this method had a good linear relationship,and the sensitivity results showed that the minimum detection limit of fluorescence quantitative PCR was 2.45×10^(2) copies/μL,which was 100 times more sensitive than 2.45×10^(4) copies/μL.This method showed no cross-reaction to Brucella,Chlamydia,and Mycoplasma.The results of intra-and inter-batch tests were stable,with the coefficient of variation less than 3%,indicating that the method had good specificity and was reproducible.The method was used to test 126 collected samples and showed a positive rate of 9.52%,compared to 7.14%by conventional PCR,with a compliance rate of 90.47%.This method provides technical support for the rapid diagnosis and prevention of clinical diseases.
作者
张锐铮
于皓同
王凯茸
张琪
张淑霞
许信刚
ZHANG Rui-zheng;YU Hao-tong;WANG Kai-rong;ZHANG Qi;ZHANG Shu-xia;XU Xin-gang(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi,712100,China)
出处
《动物医学进展》
北大核心
2024年第7期6-10,共5页
Progress In Veterinary Medicine
基金
国家重点研发计划项目(2021YFD1600704)
陕西省农业专项资金项目项目畜牧专项(XNDY2210)。
作者简介
张锐铮(1997-),男,河南汝州人,硕士研究生,主要从事兽医病原学研究;通讯作者:张淑霞;通讯作者:许信刚。
