大肠杆菌O157∶H7微滴数字PCR定量方法的建立  

Development of Droplet Digital Polymerase Chain Reaction for Quantifying Escherichia Coli O157∶H7

作　　者：董莲华[1] 张玲[1] 姜君[2] 王江南 王晶[1] 陈唯军[2] 

机构地区：[1]中国计量科学研究院,北京100029 [2]中国科学院北京基因组研究所基因组科学与信息重点实验室,北京100029 [3]北京市计量检测科学研究院北京100029

出　　处：《分析化学》2015年第3期319-324,共6页Chinese Journal of Analytical Chemistry

基　　金：中国计量科学研究院基本科研业务费资助项目(Nos.AKY1323-14,AKY1408-14)

摘　　要：以大肠杆菌O157∶H7（E.coli O157∶H7）rfb E基因为靶基因,建立了可对其准确定量的微滴数字PCR（dd PCR）方法。对dd PCR反应中的探针浓度进行了优化,考察了方法的线性范围、精密度、定量限和检出限。最终确定dd PCR反应中的最佳探针浓度为300 nmol/L。E.coli O157∶H7基因组DNA浓度范围为4-1.25×105拷贝/20μL dd PCR反应液时,dd PCR方法线性相关系数（R2）为0.999。当DNA浓度为760-88400拷贝/20μL时,方法的精密度最好（RSD〈5%）。本方法的定量限为4拷贝/20μL,检出限为3拷贝/20μL。特异性验证结果表明,建立的dd PCR方法特异性良好,对13份猪肉、牛肉和鸡肉样品的检测结果与定量PCR方法检出结果一致。A droplet digital polymerase chain reaction（ dd PCR） method for quantifying E. coli O157∶ H7 by targeting rfb E gene was developed. The probe concentration in dd PCR was optimized and the linearity range,precision,limit of detection（ LOD） and limit of quantification（ LOQ） were also evaluated. The optimized probe concentration was 300 nmol / L. The dd PCR response was linear over the E. coli O157∶ H7 genome DNA concentration range from 4 to 1. 25 ×10^5copies in 20 μL dd PCR system and the linear correlation coefficient（ R2） was 0. 999. The dd PCR precision（ RSD） was less than 5% over the DNA concentration range from 760 to 88400 copies /20 μL. The LOD and LOQ was 3 copies in 20 μL and 4 copies in 20 μL,respectively.Specificity test showed that the dd PCR was specific for detecting E. coli O157∶ H7. Both dd PCR and standard real time quantitative PCR showed the same results for 16 real samples of chicken meat,pork and beef,which indicated that dd PCR method was suitable for detection of E. coli O157∶ H7 in food.

关 键 词：微滴数字聚合酶链式反应 大肠杆菌O157∶H7 拷贝数 

分 类 号：O655.2[理学—分析化学]