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

Rapid detection of Candida albicans by real-time fluorescence quantitative polymerase chain reaction using TaqMan-minor groove binder probe and analysis of antifungal susceptibility

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。

Objective： To develop a TaqMan-minor groove binder （ TaqMan-MGB ） probe based real-time fluorescence quantitative polymerase chain reaction （ RTFQ-PCR ） assay that can rapidly quantify, detect and identify Candida albicans from clinical specimen, and analyze antifungal susceptibility. Method： Primers and probes specific to internal transcribed spacer （ ITS ） and 26S ribosomal RNA （ 26S rRNA ） genes of Candida albicans were designed. Standard plasmid containing the sequence of ITS and 26S rRNA genes were constructed for quantitative analysis. A TaqMan-MGB probe based RTFQ-PCR assay was developed. The sensitivity, specificity, reproducibility and stability of the assay were assessed. Then, the developed TaqMan-MGB probe based RTFQ- PCR assay was applied to detecting Candida albicans in 1 185 clinical specimens, and fungal culture, conventional PCR, gene cloning and sequencing were concurrently performed. Antifungal susceptibility test was done on all fungi isolates. Results： It is shown that the developed TaqMan-MGB probe based RTFQ-PCR assay could accurately detect Candida albican with 100% specificity, and other Candida species, non-Candida species, bacteria, parasites and viruses had no cross reaction. The technology was demonstrated to be highly sensitive, allowing a precise Candida albicans DNA quantitation over a range of eleven orders of magnitude, and the limit of detection （ LOD ） of the assay was determined to be 5 Colony-Forming Units （ CFU ） for Candida albicans. The reproducibility of this method was excellent with the relative standard deviation （ RSD ） within and between groups of less than 2 percent. Correlation coefficient, slope and efficiency did not vary significantly among the tests, suggesting good stability and high precision of the method. The TaqMan-MGB probe based RTFQ-PCR assay was successfully applied to quantitative detection of Candida albicans genomic load in clinical specimens, and was confirmed using conventional PCR and gene cloning and sequence analysis. The assay described in this report generates complete result in 2 h and can be used as a quantitative analytical and fast diagnostic method. The TaqMan-MGB probe based RTFQ-PCR assay was used to detect Candida albicans in ! 185 clinical specimens, in which 94 specimens were positive, while only eight specimens were positive for Candida albican obtained by fungal isolation method. The determination of the antifungal sensitivity showed that these Candida albicans isolated were susceptible to nystatin, miconazole, clotrimazole, econazole, fluconazole and voriconazole. However, their resistance to amphotericin B, ketoconazole, terbinafine and fluorocytosine were found. Conclusion： The novel TaqMan-MGB probe based RTFQ-PCR developed and evaluated in this study is considered as a rapid and simple, accurate and stable, specific and sensitive method for quantitative determination and identification of Candida albicans, and it is suitable for Candida albicans detection in animal origin products, for safety inspection of food, drug, biological product and medical instrument, and for environmental monitoring and epidemiology investigation.