重组酶聚合酶扩增在铜绿假单胞菌检测中的应用

Application of recombinase polymerase amplification in the detection of Pseudomonas aeruginosa

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，铜绿假单胞菌浓度为1×10^1-1×10^7 CFU/mL时，均能检测到阳性扩增信号。RPA中，铜绿假单胞菌浓度为1×10^1 CFU/mL时，其阳性扩增信号检出率为0；浓度为1×10^2 CFU/mL时，其阳性扩增信号检出率为67%；浓度为1×10^3-1×10^7 CFU/mL时，其阳性扩增信号检出率为100%。（3）RPA、PCR和实时荧光定量PCR中，铜绿假单胞菌出现阳性扩增信号和凝胶阳性结果，鲍氏不动杆菌、金黄色葡萄球菌、白色念珠菌及恶臭假单胞菌4种阴性对照菌未出现阳性扩增信号和凝胶阳性结果。（4）RPA中，28株甘油保存的铜绿假单胞菌临床菌株及1株棉拭子取材的铜绿假单胞菌临床菌株均出现阳性扩增信号，恶臭假单胞菌未出现阳性扩增信号；29株临床铜绿假单胞菌阳性扩增信号检出率为100%。结论 本研究建立的优化的铜绿假单胞菌RPA快速检测方法，耗时短，灵敏性及特异性强，对临床快速检测铜绿假单胞菌感染具有重要应用价值。

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 minutes, and the total time was 138 minutes. In real-time fluorescence quantitative PCR, amplification and detection could be completed simultaneously, which took 90 minutes, and the total time was 110 minutes. In RPA, amplification and detection could also be completed simultaneously, which took 15 minutes, and the total time was 35 minutes. （2） Pseudomonas putida did not show positive amplification signals or gel positive results in any of the three detection methods. The detection limit of Pseudomonas aeruginosa in real-time fluorescence quantitative PCR and PCR was 1×10^1 CFU/mL, and that of Pseudomonas aeruginosa in RPA was 1×10^2 CFU/mL. In RPA and real-time fluorescence quantitative PCR, the higher the concentration of Pseudomonas aeruginosa, the shorter threshold time and smaller the number of cycles, namely shorter time for detecting the positive amplified signal. In real-time fluorescence quantitative PCR, all positive amplification signal could be detected when the concentration of Pseudomonas aeruginosa was 1×10^1-1×10^7 CFU/mL. In RPA, the detection rate of positive amplification signal was 0 when the concentration of Pseudomonas aeruginosa was 1×10^1 CFU/mL, while the detection rate of positive amplification signal was 67% when the concentration of Pseudomonas aeruginosa was 1×10^2 CFU/mL, and the detection rate of positive amplification signal was 100% when the concentration of Pseudomonas aeruginosa was 1×10^3-1×10^7 CFU/mL. （3） In RPA, PCR, and real-time fluorescence quantitative PCR, Pseudomonas aeruginosa showed positive amplification signals and gel positive results, but there were no positive amplification signals or gel positive results in four negative control strains of Acinetobacter baumannii, Staphylococcus aureus, Candida albicans, and Pseudomonas putida. （4） In RPA, 28 clinical strains of Pseudomonas aeruginosa preserved in glycerin and 1 clinical strain of Pseudomonas aeruginosa taken by cotton swab showed positive amplification signals, while Pseudomonas putida did not show positive amplification signal. The detection rate of positive amplification signal of 29 clinical strains of Pseudomonas aeruginosa in RPA was 100%.Conclusions The established optimized RPA technology for fast detection of Pseudomonas aeruginosa requires shorter time, with high sensitivity and specificity. It was of great value in fast detection of Pseudomonas aeruginosa infection in clinic.