白色念珠菌TaqMan-小沟结合物探针实时荧光定量聚合酶链式反应快速检测及抗真菌药物敏感性分析

发布时间：2018-04-09 08:15

本文选题：白色念珠菌　切入点：内转录间隔区　出处：《药物分析杂志》2017年06期

【摘要】：目的:建立TaqMan-小沟结合物探针实时荧光定量聚合酶链式反应方法,快速定量检定临床标本中的白色念珠菌并进行抗真菌药物敏感性分析。方法:针对白色念珠菌内转录间隔区和26S核糖体核糖核酸基因设计特异性引物和探针,构建含有上述基因的标准质粒进行定量分析,建立TaqMan-小沟结合物探针实时荧光定量聚合酶链式反应检测方法,评价其特异性、灵敏度、重复性和稳定性。对1 185份临床标本中的白色念珠菌进行检测,同时进行真菌培养、普通PCR、基因克隆和测序鉴定。对所有真菌分离株进行抗真菌药物敏感性试验。结果:研究结果显示,建立的白色念珠菌TaqMan-小沟结合物探针实时荧光定量聚合酶链式反应检测方法专属性强,能准确检出白色念珠菌,而与其他念珠菌属真菌、非念珠菌属真菌、细菌、寄生虫和病毒均无交叉反应,特异性为100%。该技术灵敏度高,能精确定量检测白色念珠菌DNA线性范围达11个数量级,白色念珠菌最低检测限度为5个菌落形成单位。该方法重复性非常好,组内和组间相对标准偏差均小于2%。测试中相关系数、斜率和效率没有显著变化,表明该方法稳定性好,精密度高。将其成功应用于临床标本中白色念珠菌载量的定量检测,用普通PCR和基因克隆测序分析进行了确证。整个检测过程可在2 h内完成,可以用作定量分析快速诊断方法。应用TaqMan-小沟结合物探针实时荧光定量聚合酶链式反应从1 185份临床标本中检出94份白色念珠菌阳性标本,真菌培养获得8株存活的白色念珠菌,抗真菌药物敏感性试验分析结果显示:这些白色念珠菌分离株对制霉菌素、咪康唑、克霉唑、益康唑、氟康唑、沃尔康唑均敏感,但对两性霉素B、酮康唑、特比萘芬、氟胞嘧啶已经产生了耐药性。结论:本研究新开发评价的TaqMan-小沟结合物探针实时荧光定量聚合酶链式反应,可以快速简易,精准稳定,特异灵敏地定量检定白色念珠菌,适用于动物源性产品中白色念珠菌检测、食品药品生物制品医疗器械安全检查、环境监测、流行病学调查。
[Abstract]:Objective: to establish a real-time fluorescent quantitative polymerase chain reaction method with Taq Man-small groove conjugate probe to detect Candida albicans in clinical samples and to analyze their susceptibility to antifungal drugs.Methods: specific primers and probes were designed for the internal transcription spacer and 26s ribonucleic acid gene of Candida albicans, and the standard plasmid containing the above genes was constructed for quantitative analysis.A real-time fluorescent quantitative polymerase chain reaction method for the detection of TaqMan-small groove conjugate probe was established to evaluate its specificity, sensitivity, reproducibility and stability.Candida albicans were detected in 1 185 clinical specimens, fungi culture, common PCR, gene cloning and sequencing were performed.The antimicrobial susceptibility test was carried out on all fungi isolates.Results: the results showed that the established real-time fluorescent quantitative polymerase chain reaction method for the detection of Candida albicans TaqMan-small groove conjugate probe was highly specific and could accurately detect Candida albicans, but with other Candida fungi.Noncandida fungi, bacteria, parasites and viruses did not cross reaction, the specificity was 100.The sensitivity of this technique is high, and the linear range of accurate quantitative detection of Candida albicans DNA is up to 11 orders of magnitude, and the minimum detection limit of Candida albicans is 5 colony forming units.The reproducibility of the method was very good, and the relative standard deviations within and between groups were less than 2.The correlation coefficient, slope and efficiency are not significantly changed in the test, which indicates that the method has good stability and high precision.It was successfully applied to the quantitative detection of Candida albicans in clinical specimens and confirmed by PCR and gene clone sequencing.The whole detection process can be completed within 2 hours and can be used as a rapid diagnostic method for quantitative analysis.94 Candida albicans positive specimens were detected from 1 185 clinical specimens by real time fluorescence quantitative polymerase chain reaction with TaqMan-small groove conjugate probe. Eight strains of Candida albicans were isolated from fungal culture.The results of antimicrobial susceptibility test showed that the isolates were sensitive to nystatin, miconazole, clotrimazole, econazole, fluconazole, and Wolconazole, but were sensitive to amphotericin B, ketoconazole, terbinafine, amphotericin B, ketoconazole, terbinafine, amphotericin B, ketoconazole and terbinafine.Fluorocytosine has developed resistance.Conclusion: the Taq Man-small groove conjugate probe developed in this study can be used to detect Candida albicans in a rapid, simple, accurate and sensitive manner.It is suitable for the detection of Candida albicans in animal products, safety inspection of medical instruments for food, drug and biological products, environmental monitoring and epidemiological investigation.
【作者单位】：中国食品药品检定研究院;
【基金】：国家科技支撑计划(2013BAK11B01)资助项目
【分类号】：R440;R519

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