摘要目的 探讨实时荧光重组酶聚合酶扩增(RPA)在白色念珠菌检测中的初步应用效果.方法 (1)取1 mL白色念珠菌标准株及阴性对照菌铜绿假单胞菌、金黄色葡萄球菌、鲍氏不动杆菌、大肠杆菌、光滑念珠菌标准株菌液,使用酵母/细菌基因组试剂盒提取DNA.分析PCR、实时荧光定量PCR、实时荧光RPA检测白色念珠菌的特异性.(2)取1株白色念珠菌标准株,以光滑念珠菌为阴性对照菌,复苏摇菌并计数,10倍倍比稀释为1×107～1×101集落形成单位(CFU)/mL,同实验(1)提取DNA,分析PCR、实时荧光定量PCR、实时荧光RPA检测白色念珠球菌的灵敏性.统计实时荧光定量PCR扩增曲线达到阈值所需循环数及实时荧光RPA出现特异性扩增曲线所需时间,并与PCR结果进行比对.统计3种方法对白色念珠菌的检出下限及检出率,分析实时荧光RPA中白色念珠菌浓度与检测时间之间的相关性.(3)取1株白色念珠菌标准株,同实验(1)提取DNA,分别行PCR、实时荧光定量PCR、实时荧光RPA,统计3种方法检测总时长.(4)取31份疑似感染白色念珠菌的临床样本和1份棉拭子取材的白色念珠菌临床样本提取DNA,进行PCR与实时荧光RPA,统计阳性检出率.分别采用酵母/细菌基因组试剂盒、chelex-100加热煮沸法、液氮反复冻融法提取上述2种方法检测均出现阳性结果的临床样本的DNA.同前行实时荧光RPA检测(以光滑念珠菌为阴性对照菌)和PCR检测,阴性对照菌同实验(1),观察2种方法检测是否出现阳性结果,并统计实时荧光RPA中扩增曲线达到荧光阈值所需时间.对数据行线性相关分析和t检验. 结果 (1)3种方法检测中白色念珠菌均出现阳性结果,5种阴性对照菌均未出现阳性结果.(2)白色念珠菌菌液浓度越低,实时荧光定量PCR中扩增曲线达到阈值所需循环数越多,实时荧光RPA中出现特异性扩增曲线所需时间越长,PCR中凝胶条带的亮度越弱,3种检测方法中阴性对照菌均未出现相应的阳性结果.实时荧光RPA、PCR与实时荧光定量PCR中白色念珠菌的检出下限均为1×101 CFU/mL.实时荧光RPA中白色念珠菌浓度与检测时间呈明显负相关,r=-0.95,P＜0.01.PCR与实时荧光定量PCR对1×101～1×107 CFU/mL的白色念珠菌的阳性检出率均为100％.实时荧光RPA对1×101 CFU/mL的白色念珠菌的阳性检出率为78％,对1×102 ～1×107 CFU/mL的白色念珠菌的阳性检出率均为100％.(3)PCR、实时荧光定量PCR、实时荧光RPA检测白色念珠菌的总时间分别为133、93、35 min.(4)实时荧光RPA对31份疑似感染白色念珠菌临床样本的阳性检出率为32.26％(10/31),低于PCR的35.48％(11/31).11份临床样本经实时荧光RPA与PCR检测均出现阳性结果,阴性对照菌均未出现阳性结果.采用酵母/细菌基因组提取试剂盒、chelex-100加热煮沸法提取DNA进行实时荧光RPA,扩增曲线达到阈值所需时间分别为(438±13)、(462±12)s,二者相近(t=1.32,P＞0.05).液氮反复冻融法提取DNA进行实时荧光RPA,扩增曲线达到阈值所需时间为(584±15)s,明显长于另外2种方法(t =7.55、6.39,P＜0.01). 结论 实时荧光RPA检测白色念珠菌具有检测快速、操作简单、灵敏性高、特异性好等优点,值得临床推广应用.

abstractsObjective To explore the preliminay application effect of real-time fluorescence recombinase polymerase amplification (RPA) in the detection of Candida albicans.Methods (1) Candida albicans standard strain and negative control bacteria of Pseudomonas aeruginosa,Staphylococcus aureus,Acinetobacter baumannii,Escherichia coli,Candida glabrata 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 Candida albicans were analyzed.(2) One Candida albicans standard strain and one negative control bacteria of Candida glabrata standard strain were collected,resuscitated,and counted.Candida albicans was diluted 10 times to 1 × 107 to 1 × 101 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 Candida albicans 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 Candida albicans were analyzed,and the correlation between concentration of Candida albicans in real-time fluorescence RPA and detection time was analyzed.(3) One standard strain of Candida albicans 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 Candida albicans infection and 1 clinical sample of Candida albicans 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 Candida glabrata 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 realtime fluorescence RPA was recorded,respectively.Data were processed with linear correlation analysis and t test.Results (1) Three methods showed positive results in detecting standard strain of Candida albicans,and none of the 5 negative control bacteria showed positive results.(2) As the concentration of bacterial solution of Candida albicans 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 Candida albicans in real-time fluorescence RPA,PCR,and real-time fluorescent quantitative PCR was 1 × 101 CFU/mL.There was a significant negative correlation between the concentration of Candida albicans and the detection time in real-time fluorescence RPA (r =-0.95,P ＜ 0.01).The positive detection rates of PCR and real-time fluorescent quantitative PCR for Candida albicans of 1 × 101 to 1 × 107 CFU/mL were 100％.The positive detection rate of real-time fluorescence RPA for Candida albicans of 1 × 101 CFU/mL was 78％,and the positive detection rate of real-time fluorescence RPA for Candida albicans of 1 × 102 to 1 × 107 CFU/mL was 100％.(3) The total time of PCR,real-time fluorescent quantitative PCR,and real-time fluorescence RPA detection for Candida albicans was 133,93,and 35 min,respectively.(4) The positive detection rate of real-time fluorescence RPA for 31 clinical samples of suspected Candida albicans 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 chelex100 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(t =1.32,P ＞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 (t =7.55,6.39,P ＜ 0.01).Conclusions Real-time fluorescence RPA has advantages of rapid detection,simple operation,high sensitivity,and good specificity in detecting Candida albicans,which is worthy of clinical application.