PCR-反向微孔板杂交法检测医学真菌初步应用  

Application of PCR-reverse microplate hybridization assay for detecting and identifying fungi

作　　者：郭梅 肖林 李秀全 牟兆钦[3] 

机构地区：[1]解放军第二五四医院检验科,天津300142 [2]解放军第二七二医院检验科,天津300020 [3]军事医学科学院附属医院检验科,北京100070

出　　处：《国际检验医学杂志》2007年第10期890-893,共4页International Journal of Laboratory Medicine

摘　　要：目的通过建立PCR-反向微孔板杂交法鉴定临床常见的致病真菌，为临床治疗提供可靠依据。方法应用通用引物对真菌ERG11基因的保守序列进行扩增，扩增片段包被于微孔板中，再将真菌特异性引物与之杂交，经漂洗后显色，读取450nm处吸光度值判断结果。结果8种真菌特异性探针不与其他种的真菌、细菌和人类DNA分子杂交，而只和其对应的真菌DNA扩增产物反应，呈现阳性杂交结果。55例临床血液病、肿瘤等患者的血液、脑脊液及痰等标本分别用PCR一反向微孔板杂交法和真菌培养法（API20C）鉴定，2种方法的结果基本一致（阳性率分别为34．5％和30．9％）。结论PCR-反向微孔板杂交法可用于临床不同标本致病真菌的检测。Objective The aim of the study is to develop PCR-reverse microplate hybridization assay for detecting and identifying common fungi in clinic, so as to provide a basis for clinical therapy. Methods A highly conserved region of the ERGll gene was amplified by applying the consensus primer. The amplified fragment was coated into microplate wells, and hybridized with the fungipecific probe. After rinsed, the samples were colourated by addition of the colorimetric substrate. The optical densities at 450 nm were read. Results Eight fungi species-specific probes were only hybridized with their respective PCR products. No cross-hybridization was detected with any other fungi, bacterial and human DNA tested. 55 cases of different clinical samples, including blood, cerebrospinal fluid and sputum from patients with hematonosis, tumor or radiation pneumonia were detected by applying PCR-reverse microplate hybridization assay and conventional culture （API 20C） simultaneously. The results showed concordant （positive rate were 34.5% and 30.9% respectively）. Conclusion PCR-reverse microplate hybridization assay is a rapid and convenient, and available for detecting and identifying common fungi in clinic.

关 键 词：真菌 PCR 反向微孔板杂交 

分 类 号：R446[医药卫生—诊断学] R379[医药卫生—临床医学]