依据 GenBank 中大肠杆菌、肺炎克雷伯氏菌和不动杆菌的部分已知序列，选取大肠杆菌的uidA基因、肺炎克雷伯氏菌的khe基因和不动杆菌的secE基因，利用Primer Premier 5设计引物，选出扩增序列的3对引物及相应的Taqman探针，uidA、khe、secE探针5'端分别标记FAM、HEX、CY5荧光发射基因，3'端标记BHQ1淬灭荧光基因。通过优化反应条件，建立一种同时检测大肠杆菌、肺炎克雷伯氏菌和不动杆菌的诊断方法。特异性和敏感性试验显示，该方法能特异地检测大肠杆菌、肺炎克雷伯氏菌和不动杆菌，其最低检测限分别为2×10-1 CFU/μL、9.2×10-1 CFU/μL和4.3×10-1 CFU /μL。整个检测扩增在2小时内完成。该结果表明本试验所建立的三重荧光定量PCR方法的灵敏度、重复性及特异性均较好，可用于同时快速检测三种病原菌。
According to the partially known sequences of Escherichia coli，Klebsiella pneumoniae and Acinetobacter in GenBank，the uidA gene of Escherichia coli，khe gene of Klebsiella pneumoniae and secE gene of Acinetobacter were selected. Then 3 pairs of primers and Taqman probes of which 5' end were labeled with fluorophores（FAM，HEX，CY5）respectively ，and 3' end marked with BHQ1 quenchers were designed by Primer Premier 5. By optimizing the reaction conditions，a diagnostic method which could detect Escherichia coli，Klebsiella pneumoniae and Acinetobacter simultaneously was established. Experiments of specificity and sensitivity showed that this method could specifically detect the above three genes，while the lowest detection limits were 2×10-1 CFU/μL，9.2×10-1 CFU/μL and 4.3×10-1 CFU /μL，respectively. The whole amplification was completed within two hours. In conclusion，a triple quantitative PCR with good sensitivity，reproducibility and specificity was established ，it could be used for rapid and simultaneous detection of the three pathogens.