摘要
目的 建立一种优化的重组酶聚合酶扩增（RPA）方法，用于临床快速检测铜绿假单胞菌。方法 （1）提取1株铜绿假单胞菌标准菌株DNA模板，采用PCR、实时荧光定量PCR和RPA进行检测，统计3种方法检测的加样时长、扩增时长和检测时长。（2）取1株铜绿假单胞菌标准菌株，复苏摇菌后逐次稀释为1×10^7、1×10^6、1×10^5、1×10^4、1×10^3、1×10^2、1×10^1集落形成单位（CFU）/mL 7个浓度梯度，以恶臭假单胞菌为阴性对照，分别提取DNA模板，进行RPA、实时荧光定量PCR及PCR，分析3种方法检测铜绿假单胞菌的灵敏性。（3）取1株铜绿假单胞菌标准菌株和4株阴性对照菌（金黄色葡萄球菌、鲍氏不动杆菌、白色念珠菌和恶臭假单胞菌），分别提取DNA模板，行RPA、实时荧光定量PCR及PCR，分析3种方法检测铜绿假单胞菌的特异性。（4）取28株甘油保存的铜绿假单胞菌临床菌株、1株棉拭子取材的铜绿假单胞菌临床菌株，以恶臭假单胞菌为阴性对照，分别提取DNA模板，行RPA。观察上述菌株是否出现阳性扩增信号，并计算其检出率。所有实验重复3次。采用GraphPad Prism 5.01统计软件对灵敏性结果进行分析。结果 （1）RPA、PCR和实时荧光定量PCR检测铜绿假单胞菌的加样时间均为20 min，其中PCR扩增98 min，凝胶检测20 min，共计138 min；实时荧光定量PCR扩增和检测可同步完成，需90 min，共计110 min；RPA中扩增和检测也可同步完成，需15 min，共计35 min。（2）3种检测方法中恶臭假单胞菌均未出现阳性扩增信号和凝胶阳性结果。实时荧光定量PCR和PCR中铜绿假单胞菌检测下限为1×10^1 CFU/mL，RPA中铜绿假单胞菌检测下限为1×10^2 CFU/mL。RPA、实时荧光定量PCR中，铜绿假单胞菌浓度越高，出现阈值时间越短、循环数越少，即检测到阳性扩增信号的时间越短。实时荧光定量PCR中，铜绿假单胞菌浓�
Objective To establish an optimized method of recombinase polymerase amplification （RPA） to rapidly detect Pseudomonas aeruginosa in clinic.Methods （1） The DNA templates of one standard Pseudomonas aeruginosa strain was extracted and detected by polymerase chain reaction （PCR）, real-time fluorescence quantitative PCR and RPA. Time of sample loading, time of amplification, and time of detection of the three methods were recorded. （2） One standard Pseudomonas aeruginosa strain was diluted in 7 concentrations of 1×10^7, 1×106, 1×105, 1×104, 1×10^3, 1×10^2, and 1×10^1 colony forming unit （CFU）/mL after recovery and cultivation. The DNA templates of Pseudomonas aeruginosa and negative control strain Pseudomonas putida were extracted and detected by PCR, real-time fluorescence quantitative PCR, and RPA separately. The sensitivity of the three methods in detecting Pseudomonas aeruginosa was analyzed. （3） The DNA templates of one standard Pseudomonas aeruginosa strain and four negative control strains （Staphylococcus aureus, Acinetobacter baumanii, Candida albicans, and Pseudomonas putida） were extracted separately, and then they were detected by PCR, real-time fluorescence quantitative PCR, and RPA. The specificity of the three methods in detecting Pseudomonas aeruginosa was analyzed. （4） The DNA templates of 28 clinical strains of Pseudomonas aeruginosa preserved in glycerin, 1 clinical strain of which was taken by cotton swab, and negative control strain Pseudomonas putida were extracted separately, and then they were detected by RPA. Positive amplification signals of the clinical strains were observed, and the detection rate was calculated. All experiments were repeated for 3 times. Sensitivity results were analyzed by GraphPad Prism 5.01 statistical software.Results （1） The loading time of RPA, PCR, and real-time fluorescence quantitative PCR for detecting Pseudomonas aeruginosa were all 20 minutes. In PCR, time of amplification was 98 minutes, time of gel detection was 20
作者
金晓君
龚雅利
杨莉
莫邦辉
彭毅志
何鹏
赵军宁
李晓鲁
Jin Xiaojun;Gong Yali;Yang Li;Mo Banghui;Peng Yizhi;He Peng;Zhao Junning;Li Xiaolu(Department of Burns and Plastic Surgery, the First Affiliated Hospital of Southwest Medical University, Luzhou 646000, Chin)
出处
《中华烧伤杂志》
CAS
CSCD
北大核心
2018年第4期233-239,共7页
Chinese Journal of Burns
基金
国家自然科学基金（81171780、30600648）
西南医院军事医学与战创伤救治临床新技术计划（SWH2016YSCXYB-10）
关键词
假单胞菌
铜绿
重组酶聚合酶扩增
恒温扩增
DNA快速检测
Pseudomonas aeruginosa
Recombinase polymerase amplification
Isothermal amplification
DNA rapid detection
