检测阴沟肠杆菌的数字PCR定量方法建立

Establishment of the digital PCR assay for detection of Enterobacter cloacae

以阴沟肠杆菌Amp C酶基因为靶基因,建立了可对其准确定量的数字PCR(dd PCR)方法。对dd PCR反应中的探针浓度进行了优化,并将该方法与实时荧光PCR方法做对比来检测阴沟肠杆菌。结果表明,最终确定dd PCR反应中的最佳探针浓度为400 nmol/L,该方法检测阴沟肠杆菌的最低DNA拷贝数为8.9拷贝/μL,且重复性良好。表明数字PCR方法灵敏度高、特异性好,可用于阴沟肠杆菌的鉴定。

A droplet digital polymerase chain reaction（ dd PCR） method for quantifying Enterobacter cloacae based on amp C gene was developed. The method was compared with real- time fluorescence PCR method and the probe concentration in dd PCR was optimized. The results showed that the optimized probe concentration was 400 nmol / L. The minimum DNA copies of E. cloacae was 8. 9 copies / μL by the dd PCR method and the detection result was highly reproducible. It suggests that the dd PCR method established in this study is sensitive and specific,and can be used for the identification of E. cloacae.