[Preliminary application of real-time fluorescence recombinase polymerase amplification in ].

To explore the preliminary application effect of real-time fluorescence recombinase polymerase amplification (RPA) in the detection of . (1) standard strain and negative control bacteria of standard strains of respectively 1 mL were collected and their DNA were extracted by yeast/bacterial genomic kit. The specificity of polymerase chain reaction (PCR), real-time fluorescent quantitative PCR, and real-time fluorescence RPA in detecting were analyzed. (2) One standard strain and one negative control bacteria of standard strain were collected, resuscitated, and counted. was diluted 10 times to 1×10(7) to 1×10(1) colony-forming unit (CFU)/mL. The DNA of the two bacteria were extracted as experiment (1). The sensitivity of PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA in detecting were analyzed. The number of cycles for amplification curve to reach the threshold in real-time fluorescent quantitative PCR, and time of appearance of specific amplification curve in real-time fluorescence RPA were recorded and compared with the results in PCR. The detection limit and rate of the above-mentioned 3 methods in detecting were analyzed, and the correlation between concentration of in real-time fluorescence RPA and detection time was analyzed. (3) One standard strain of was collected, and the DNA was extracted as experiment (1) and detected by PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA. The total detection time of the above-mentioned 3 methods was recorded, respectively. (4) The DNA of 31 clinical samples of suspected infection and 1 clinical sample of collected from cotton swab were extracted, PCR and real-time fluorescence RPA were carried out, and the positive detection rates of the above-mentioned methods were calculated. The DNA of the clinical samples with positive results in both PCR and real-time fluorescence RPA were extracted by yeast/bacterial genomic kit, chelex-100 boiling method, and repeatedly freeze-thawing with liquid nitrogen method, and real-time fluorescence RPA and PCR were carried out. The negative control bacteria was in real-time fluorescence RPA, while negative control bacteria in PCR were the same as experiment (1). The positive results in PCR and real-time fluorescence RPA were observed and time for amplification curve to reach the fluorescence threshold in real-time fluorescence RPA was recorded, respectively. Data were processed with linear correlation analysis and test. (1) Three methods showed positive results in detecting standard strain of and none of the 5 negative control bacteria showed positive results. (2) As the concentration of bacterial solution of decreased, the number of cycles for the amplification curve to reach the threshold increased in real-time fluorescent quantitative PCR, the time for appearance of specific amplification curve prolonged in real-time fluorescence RPA, and brightness of the gel strip weakened in PCR. None of the negative control bacteria in the above-mentioned 3 detection methods showed corresponding positive results. The detection limit of in real-time fluorescence RPA, PCR, and real-time fluorescent quantitative PCR was 1×10(1) CFU/mL. There was a significant negative correlation between the concentration of and the detection time in real-time fluorescence RPA (=-0.95, <0.01). The positive detection rates of PCR and real-time fluorescent quantitative PCR for of 1×10(1) to 1×10(7) CFU/mL were 100%. The positive detection rate of real-time fluorescence RPA for of 1×10(1) CFU/mL was 78%, and the positive detection rate of real-time fluorescence RPA for of 1×10(2) to 1×10(7) CFU/mL was 100%. (3) The total time of PCR, real-time fluorescent quantitative PCR, and real-time fluorescence RPA detection for was 133, 93, and 35 min, respectively. (4) The positive detection rate of real-time fluorescence RPA for 31 clinical samples of suspected infection was 32.26% (10/31), which was slightly lower than 35.48% (11/31) of PCR. Eleven clinical samples showed positive results both in real-time fluorescence RPA and PCR detection. No positive result was observed in the negative control bacteria detected both by real-time fluorescence RPA and PCR. The DNA was extracted by yeast/bacterial genomic extraction kit and chelex-100 boiling method for real-time fluorescence RPA detection. The time for the amplification curve to reach the threshold was (438±13) and (462±12) s, respectively, which was close (=1.32, >0.05). The DNA was extracted by repeatedly freeze-thawing with liquid nitrogen method for real-time fluorescence RPA, and the time for the amplification curve to reach the threshold in real-time fluorescence RPA was (584±15) s, which was significantly longer than that in the other 2 methods (=7.55, 6.39, <0.01). Real-time fluorescence RPA has advantages of rapid detection, simple operation, high sensitivity, and good specificity in detecting which is worthy of clinical application.