实时荧光定量PCR快速检测细菌对抗菌素敏感性与耐药性表型的方法建立  

Establishment of real-time fluorescent quantitative PCR for rapid detection of bacteria antibiotic susceptibility and resistance phenotype

作　　者：谢思露 向瑶[1] 杨朝国[1] XIE Si-lu;XIANG Yao;YANG Chao-guo(School of Medical Technology,Chengdu University of Traditional Chinese Medicine,Chengdu,Sichuan 610075,China)

机构地区：[1]成都中医药大学医学技术学院,四川成都610075

出　　处：《现代预防医学》2019年第9期1684-1688,共5页Modern Preventive Medicine

摘　　要：目的建立快速检测细菌对抗菌素的敏感性与耐药性表型的实时荧光定量PCR(qPCR)方法。方法用基于细菌16S rRNA基因通用引物和通用探针的qPCR检测大肠埃希菌、金黄色葡萄球菌和铜绿假单胞菌临床分离菌株基因组DNA,绘制生长曲线以确定细菌显著生长的最短培养时间。计算细菌在含和不含抗菌素的M-H肉汤中的相对生长率,以VITEK 2 Compact全自动细菌分析系统的药敏结果为金标准,对细菌相对生长率进行ROC曲线分析,确定区分细菌对抗菌素敏感与耐药的相对生长率分界值。用20株肠杆菌科细菌和10株革兰阳性球菌临床分离菌株验证方法的可靠性。结果细菌显著生长的最短培养时间为3小时。区分大肠埃希菌、金黄色葡萄球菌和铜绿假单胞菌对抗菌素敏感与耐药的相对生长率分界值分别为0.44、0.40和0.48本方法检测30株大肠埃希菌对3种抗菌素的敏感性和耐药性的准确度分别为98.4%和100.0%,30株金黄色葡萄球菌对3种抗菌素的敏感性和耐药性的准确度均为100.0%,30株铜绿假单胞菌对3种抗菌素敏感性和耐药性的准确度分别为60.0%和64.0%。20株肠杆菌科细菌和10株革兰阳性球菌对抗菌素的敏感性与耐药性结果的总准确度分别为92.7%和93.3%。结论本方法能快速准确为临床提供肠杆菌科细菌和革兰阳性球菌对抗菌素的敏感性与耐药性表型,但对非发酵菌药敏结果的准确性不能满足临床要求。Objective To establish a real - time fluorescence quantitative PCR ( qPCR) assay for rapidly detecting the antibiotic susceptibility and resistance phenotype of bacteria. Methods The genomic DNA of clinical isolates of Escherichia coli,Staphylococcus aureus and Pseudomonas aeruginosa was detected by qPCR based on universal primers and universal probes of bacterial 16S rRNA gene,and the growth curves were drawn to determine the minimum culture time for significant bacterial growth. Relative growth rates of bacteria in M - H broth with and without antibiotics were calculated. Taking the drug susceptibility results of VITEK 2 Compact automatic bacterial analysis system as the gold standard,ROC analysis of the relative growth rate of the bacteria was conducted to determine the cut off value of bacteria antibiotic sensitivity and resistance. The reliability of the method was verified with clinical isolates of 20 strains of Enterobacteriaceae and 10 strains of gram - positive cocci. Results The minimum culture time for significant bacterial growth was 3 hours. For Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa,the relative growth rate cut off values of antibiotic susceptibility and resistance were 0. 44, 0. 40 and 0. 48,respectively. The susceptibility and resistance accuracies of 30 strains of Escherichia coli of this method to the three antibiotics were 98. 4% and 100. 0%, respectively. The accuracies were both 100. 00% for 30 strains of Staphylococcus aureus. For 30 strains of Pseudomonas aeruginosa, accuracies were 60. 0% and 64. 0%, respectively. The overall accuracies of the drug susceptibility results for the 20 strains of Enterobacteriaceae and 10 strains of Gram - positive cocci were 92. 7% and 93. 3%,respectively. Conclusion This method can rapidly and accurately provide the clinical antibiotic susceptibility and drug resistance phenotype of Enterobacteriaceae and Gram - positive cocci,but the accuracy of the drug sensitivity test results of the non - fermenting bacteria can not meet the c

关 键 词：实时荧光定量聚合酶链反应 细菌 16SRRNA基因 抗菌素 敏感性 

分 类 号：R115[医药卫生—公共卫生与预防医学]