双重荧光定量聚合酶链反应检测烟曲霉菌与黑曲霉菌的临床应用  

Detection of Aspergillusfumigatusand Aspergillus niger by duplex quantitative real-time PCR assay

作　　者：毛晓露[1] 卢忠心[1] 石小燕 张文静 

机构地区：[1]武汉市中心医院检验科 [2]中心实验室 [3]血液科,湖北武汉430014

出　　处：《中华医院感染学杂志》2013年第19期4847-4849,共3页Chinese Journal of Nosocomiology

基　　金：武汉市卫生局基金(WX10C13)

摘　　要：目的建立检测烟曲霉菌和黑曲霉菌的双重荧光定量聚合酶链反应方法,探讨其临床应用价值,为快速准确地鉴定侵袭性曲霉菌寻求新的检测技术。方法设计可扩增烟曲霉菌和黑曲霉菌18sRNA引物和探针,建立双重荧光定量聚合酶链反应体系并进行方法学评价;选取45例临床确诊的侵袭性真菌感染(IF)患者和20例阴性健康对照者的全血,应用本方法对以上两种真菌进行DNA检测;结果与常规鉴定方法比较,评估该方法的敏感性和特异性。结果对于纯化后的烟曲霉菌和黑曲霉菌DNA,该方法的敏感性为1拷贝,特异性达到100%,批内和批间试验均显示了较好的重复性;临床标本检测结果显示,该方法与常规方法比较,敏感性和特异性均为100%。结论应用双重荧光定量聚合酶链反应方法同时检测烟曲霉菌和黑曲霉菌具有良好的敏感性、特异性和重复性,与常规鉴定方法结果相符,有利于烟曲霉菌和黑曲霉菌感染的早期快速诊断和实现对进展期感染者的动态监测。OBJECTIVE To set up a duplex quantitative real-time PCR （duplex QPCR） assay to detect Aspergillus fumigatus and Aspergillus niger and evaluate its clinical diagnostic value so as to explore a new method to identify invasive aspergillosis accurately and rapidly. METHODS The 18S rDNA area of high consensus sequence of Asper- gillus fumigatus and Aspergillus niger were selected to design the primers and probes and QPCR method was set up and evaluated. Whole blood of 45 invasive fungal infection patients and 20 negative controls were tested by the QPCR method, the sensitivity and specificity of which were evaluated according to the routine methods. RESULTS The sensitivity and specificity of the QPCR method were 1 copy and 100%, and the reproducibility was good. The clinical test results showed that the sensitivity and specificity were both 100~ according to the routine methods. CONCLUSION A duplex QPCR method has been successfully set up with high sensitivity, specificity and rapidity to detect A. fumigatus and A. niger. The results are consistent with routine methods. It may he more useful for the early diagnosis for A. furnigatus and A. niger infections and for dynamic monitoring of the patients at risk of infections during developing period.

关 键 词：双重荧光定量聚合酶链反应 烟曲霉菌 黑曲霉菌 

分 类 号：R446[医药卫生—诊断学]