布鲁氏菌多重荧光定量PCR方法的建立与应用  

Application of multiplex fluorescence quantitative PCR for the detection of Brucella from different samples

作　　者：刘佳音 田国忠[2] 于高娃 杜松楠 陈俊杰 孟颖 杨晓雯 范玉 赵鸿雁[2] 朴东日[2] 杨文杰 姜海 LIU Jia-yin;TIAN Guo-zhong;YU Gao-wa;DU Song-nan;CHEN Jun-jie;MENG Ying;YANG Xiao-wen;Fan Yu;ZHAO Hong-yan;PIAO Dong-ri;YANG Wen-jie;JIANG Hai(School of Public Health,Baotou Medical College Inner Mongolia University of Science and Technology,Baotou 014060,China;State Key Laboratory for Infections Disease Prevention Control,Collaborative Innovation Center for Diagnosis and Treatment of Infectious Disease,National Institute for Communicable Disease Prevention and Control,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Tongliao Center for Disease Control and prevention,Inner Mongolia Autonomous Region,Tongliao 028000,China;School of Medical Technology and Anesthesia,Baotou Medical College Inner Mongolia University of Science and Technology,Baotou 014060,China)

机构地区：[1]内蒙古科技大学包头医学院公共卫生学院,包头014060 [2]中国疾病预防控制中心传染病预防控制所,传染病预防控制国家重点实验室,感染性疾病协同诊治协同创新中心,北京102206 [3]内蒙古自治区通辽市疾病预防控制中心,通辽028000 [4]内蒙古科技大学包头医学院医学技术与麻醉学院,包头014060

出　　处：《中国人兽共患病学报》2022年第4期309-316,共8页Chinese Journal of Zoonoses

基　　金：国家重点研发计划(No.2020YFA0907101);“十三五”国家科技重大专项“艾滋病和病毒性肝炎等重大传染病防治”(No.2018ZX10201002)联合资助。

摘　　要：目的探讨应用多重荧光定量PCR方法检测待检者血液、血清以及环境样本中布鲁氏菌的价值。方法根据149名待测者的就诊情况进行分类,其中疑似组75人、治疗组74人。对收集的环境和待检者的样本应用多重荧光定量PCR方法检测,结果进行统计分析。将3对引物、探针的目的基因克隆到PUC57载体上制作阳性标准品,进行灵敏度检测和标准曲线的绘制。结果通过对149名待检者的样本进行荧光定量PCR检测,单重、双重和三重方法的阳性率分别为79.2%、34.2%和27.5%。单重与双重,单重与三重荧光定量方法的阳性率差异均有统计学意义,χ^(2)值分别为55.42和73.42,P<0.05。布鲁氏菌属的单重荧光定量PCR结果生成的标准曲线为y=-3.7732x+43.188,R^(2)=0.9953,线性关系良好,最低检测范围为101 copies/μL。经过多重荧光定量方法检测的环境样本结果全部为阳性。结论单重荧光定量PCR方法灵敏度较好,建议与血清学方法联合使用对高危人群进行定期筛查,三重荧光定量方法可以在一次实验中既能完成属水平的鉴定又能区分牛种和羊种布鲁氏菌,对于环境样品具有良好的扩增效果,2种方法对于布鲁氏菌病的预防控制具有重要作用。To explore the value of multiplex fluorescence quantitative PCR in the detection of Brucella in blood,serum and environmental samples.149 subjects were classified according to their medical conditions,including 75 in the suspected group and 74 in the treatment group.The collected samples of environment and subjects were detected by multiplex fluorescence quantitative PCR,and the results were statistically analyzed.The target genes of three pairs of primers and probes were cloned into PUC57 vector to make positive standard for sensitivity detection and standard curve drawing.The positive rates of single,double and triple methods were 79.2%,34.2%and 27.5%respectively.The positive rates of single and double,single and triple fluorescence quantitative methods were statistically different,and the chi-square values were 55.42 and 73.42,respectively(P<0.05).The standard curve generated by single fluorescence quantitative PCR of Brucella is y=-3.7732x+43.188,R^(2)=0.9953,the linear relationship is good,and the minimum detection range is 10^(1) copies/μl.All the environmental samples detected by multiple fluorescence quantitative method were positive.Multiple fluorescence quantitative PCR can be used to detect blood,serum and environmental samples.Triple fluorescence quantitative method can not only complete the identification of genus level,but also distinguish Brucella from bovine and sheep species in one experiment,but also realize rapid quantification.It plays an important role in the prevention and control of brucellosis.

关 键 词：多重荧光定量PCR 布鲁氏菌 灵敏度 

分 类 号：R378.5[医药卫生—病原生物学]