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

Aspergillosis detection by nucleic acid sequence-based amplification and molecular beacon

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。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)技术进行荧光检测,并对该方法进行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。

Objective To establish a quick,sensitive,specific and quantitative method for detecting aspergillus for clinical diagnosis of aspergillosis.Methods Aspergillus fumigates,Aspergillus flavus and Aspergillus niger were cultured,and the spores were harvested.The total RNA was extracted from the spores.Ten-fold serially titrated Aspergillus conidia（101 to 106 spores） were detected by nucleic acid sequence-based amplification（NASBA） and molecular beacon（MB） platform to establish a standard curve.The sensitivity and specificity of the method were evaluated.Results The reaction time（minute） of the fluorescence signal rising above the fixed threshold was linearly correlated with the logarithm of the spore amount（y =-10.7x ＋ 81.6,r = 0.988）.The detection limit of this method was 1 spore.The RNA extracted from aspergillus and the comparisons（Staphylococcus aureus,Escherichia coli,Pseudomonas aeruginosa,Candida albicans,Candida tropicalis and Cryptococcus neoformans） were amplified by NASBA,and were analyzed by electrophoresis.NASBA did not cross-react with the non-target bacteria or fungi.Conclusion The method of NASBA combined with MB has high sensitivity and specificity,and can be potentially used for medical diagnosis of aspergillosis.