核酸序列依赖扩增联合分子信标检测曲霉菌感染-第三军医大学学报.DOC

核酸序列依赖扩增联合分子信标对曲霉菌感染的检测效果研究 王立朋1，何云燕2，夏云1,王慧娟1（400016 重庆,1重庆医科大学附属第一医院检验科；400013 重庆,2重庆中山医院检验科，） [摘要] 目的 建立快速，灵敏，特异的定量检测曲霉菌感染的诊断方法，为下一步的临床应用打下基础。方法 以烟曲霉菌、黄曲霉菌及黑曲霉菌的孢子配制菌液，提取RNA后，采用核酸序列依赖扩增（nucleic acid sequence-based amplification，NASBA）联合分子信标（molecular beacon，MB）技术来进行荧光检测，并对该方法进行了灵敏度和特异性分析。结果 扩增过程中累计荧光量达到设定的荧光阈值所需循环数与标本中霉菌孢子量的对数存在线性相关关系（y= -10.7x+81.6，r=0.988）。将含有梯度数量霉菌孢子的标本提取总RNA后进行检测，可建立相应标准曲线。该方法的灵敏度可达到1个孢子；对照菌株（金黄色葡萄球菌、大肠埃希菌、铜绿假单胞菌、白色念珠菌、热带念珠菌、新型隐球菌）与曲霉菌提取总RNA经扩增后的产物进行电泳分析，发现仅曲霉菌出现特异性条带。结论 NASBA结合分子信标检测曲霉菌具有灵敏度高、特异性强的特点，具有潜在利用价值，有望成为曲霉菌感染的临床诊断方法。 [关键词] 核酸序列依赖扩增；分子信标；曲霉菌；诊断 [中图法分类号] R44 [文献标志码] A Detecting aspergillosis by nucleic acid sequence-based amplification and molecular beacon Wang Lipeng1,He Yunyan2,Xia Yun1,Wang Huijuan1(1 Department of Clinical Laboratory Of The First Affiliated Hospital of Chongqing Medical University;2 Department of Clinical Laboratory Of Chongqing Zhongshan Hospital) [Abstract] Objective To establish a quick, sensitive, specific and quantitative method for detecting aspergillosis for clinical diagnosis. Methods The Aspergillus fumigates，Aspergillus flavus，Aspergillus niger was cultured and the spores was harvested, then the total RNA was extracted. Ten fold serially titrated Aspergillus conidia(101 to 106 spores) were detected by nucleic acid sequence-based amplification and molecular beacon platform to establish a standard curve. The sensitivity and specitivity of this method was also evaluated. Results The reaction time(minute) when the fluorescence signal rises above the fixed threshold was linear correlated with the logarithm of the spores amount（y = -10.7x+81.6，r =0.988）. The detection limit of this method was 1 spore; RNA extracted from Aspergillus and comparisons (Staphylococcus aureus, Escherrichia coli, Pseudomonas aeruginosa, Candida albicans, Candida tropicalis， Cryptococcus neoformans) were amplificated by NASBA and then analysed by electrophoresis. NASBA do not cross react with