[Application of recombinase polymerase amplification in the detection of ].

To establish an optimized method of recombinase polymerase amplification (RPA) to rapidly detect in clinic. (1) The DNA templates of one standard 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 strain was diluted in 7 concentrations of 1×10(7,) 1×10(6,) 1×10(5,) 1×10(4,) 1×10(3,) 1×10(2,) and 1×10(1) colony forming unit (CFU)/mL after recovery and cultivation. The DNA templates of and negative control strain were extracted and detected by PCR, real-time fluorescence quantitative PCR, and RPA separately. The sensitivity of the three methods in detecting was analyzed. (3) The DNA templates of one standard strain and four negative control strains ( and ) 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 was analyzed. (4) The DNA templates of 28 clinical strains of preserved in glycerin, 1 clinical strain of which was taken by cotton swab, and negative control strain 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. (1) The loading time of RPA, PCR, and real-time fluorescence quantitative PCR for detecting 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) did not show positive amplification signals or gel positive results in any of the three detection methods. The detection limit of in real-time fluorescence quantitative PCR and PCR was 1×10(1) CFU/mL, and that of in RPA was 1×10(2) CFU/mL. In RPA and real-time fluorescence quantitative PCR, the higher the concentration of 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 was 1×10(1)-1×10(7) CFU/mL. In RPA, the detection rate of positive amplification signal was 0 when the concentration of was 1×10(1) CFU/mL, while the detection rate of positive amplification signal was 67% when the concentration of was 1×10(2) CFU/mL, and the detection rate of positive amplification signal was 100% when the concentration of was 1×10(3)-1×10(7) CFU/mL. (3) In RPA, PCR, and real-time fluorescence quantitative PCR, 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 and . (4) In RPA, 28 clinical strains of preserved in glycerin and 1 clinical strain of taken by cotton swab showed positive amplification signals, while did not show positive amplification signal. The detection rate of positive amplification signal of 29 clinical strains of in RPA was 100%. The established optimized RPA technology for fast detection of requires shorter time, with high sensitivity and specificity. It was of great value in fast detection of infection in clinic.