实时荧光定量PCR诊断侵袭性真菌病方法的建立及应用  

Quantitative real-time polymerase chain reaction for the diagnosis of invasive fungal disease

作　　者：鲁巧云[1] 余进[1] 高露娟[1] 郑罡[2] 李若瑜[1] 

机构地区：[1]北京大学第一医院皮肤性病科北京大学真菌和真菌病研究中心,100034 [2]贵阳医学院第一附属医院急诊重症监护病房

出　　处：《中华医学杂志》2012年第12期822-826,共5页National Medical Journal of China

摘　　要：目的建立侵袭性真菌病的实时荧光定量PCR诊断方法，初步评价其在侵袭性真菌病诊断中的价值。方法设计真菌通用、曲霉和念珠菌的属特异性引物和TaqMan探针，检测标准菌株和部分临床分离株，探讨这3个检测体系的敏感性和特异性。收集确诊或除外侵袭性真菌病的21例患者的血清标本，验证实时荧光定量PCR诊断方法在临床诊断中的准确性。结果3个检测体系实现了在相同PCR条件下的整合。真菌通用检测体系能检测大部分病原真菌，包括烟曲霉、黄曲霉、黑曲霉、白念珠菌、近平滑念珠菌、热带念珠菌、克柔念珠菌、光滑念珠菌、新生隐球菌、多变根毛霉、少根根霉以及马内菲青霉；曲霉检测体系能检测烟曲霉、黄曲霉和黑曲霉；念珠菌检测体系能检测白念珠菌、近平滑念珠菌和热带念珠菌。真菌通用检测体系能检测到4pg基因组DNA，曲霉和念珠茵的检测体系均能检测到2个拷贝数。11例侵袭性真菌病患者的11份血清标本经该诊断体系检测，结果与传统方法的诊断结果完全吻合，且3个检测体系之间有很好的一致性，其中2例侵袭性曲霉病和2例侵袭性白念珠菌病均被鉴定至属，其他7例患者亦可通过真菌通用检测体系被诊断为侵袭性真菌病。10例除外侵袭性真菌病患者的10份血清均无扩增。结论建立的真菌通用检测体系能检测临床常见的深部病原真菌，且曲霉和念珠菌检测体系能进一步将临床常见的曲霉和念珠菌鉴定至属的水平。Objective To establish the diagnostic assays of quantitative real-time polymerase chain reaction （PCR） for the diagnosis of invasive fungal disease and evaluate their accuracy in clinical samples. Methods Three assays have been developed for the diagnosis of clinically prevalent fungi, Aspergillus spp. and Candida spp.. Their analytical sensitivity and specificity were evaluated. Twenty-one serum samples from invasive fungal disease patients and non-invasive fungal disease patients were analyzed by the diagnostic assays and the accuracy was evaluated. Results Three assays were managed to run at the same condition. The universal fungi assay was able to detect, but not differentiate between most of the pathogenic fungi, including A. fumigatus, A. flavus, A. niger, C. albicans, C. parapsilosis, C. tropicalis, C. kruseii, C. glabrata, Cryptococcus neoformans, Rhizomucor variabilis, Rhizopus arrhizus and Penicillosis marneffei. The assays of Aspergillus spp. and Candida spp. were able to detect, but not differentiate between A. fumigatus, A. flavus, A. niger and C. albicans, C. parapsilosis, C. tropicalis. The detection limits of assays were 4 pg of A. fumigatus gDNA, 2 copies of A. fumigatus and 2 copies of C. albicans respectively. The results of quantitative real-time PCR matched well with each other and were identical to the classical procedures in all patients. Among the 11 patients with invasive fungal disease, the pathogens were identified down to a genus level in 2 invasive aspergillosis patients and 2 invasive candidiasis patients. And the other 7 patients could be diagnosed with invasive fungal disease by the universal fungi assay. All the 10 serum samples from non-invasive fungal disease patients were revealed as negative. Conclusion The universal fungi assay is helpful in screening while the assays of Aspergillus spp. and the Candida spp. may identify the pathogens down to a genus level.

关 键 词：真菌病 聚合酶链反应 诊断 

分 类 号：R440[医药卫生—诊断学]