NASBA-ELISA检测曲霉菌方法的建立及在侵袭性曲霉病诊断中的应用评价

Development and application of diagnostic NASBA- ELISA for invasive aspergillosis

摘要:

目的：建立基于核酸序列依赖扩增的酶联免疫（enzyme-linked immunosorbent assay of nucleic acid sequence-based am－plification，NASBA-ELISA）法用于侵袭性曲霉病（invasive aspergillosis，IA）的诊断，并评价其诊断价值。方法：将核酸序列依赖扩增技术（nucleic acid sequence-based amplification，NASBA）方法和高灵敏度的地高辛检测系统相结合，采用地高辛标记NASBA恒温扩增曲霉菌特异性18sRNA基因片段，用生物素标记的DNA探针在链酶亲和素包被的微孔板孔中杂交捕获地高辛标记的NASBA扩增产物，最后加入碱性磷酸酶标记的地高辛抗体和底物进行酶标显色，应用此法分别对梯度数量的曲霉菌孢子和其他临床常见菌株进行检测以评价其灵敏度和特异度，并通过对82例临床标本（32例阳性，50例阴性）的检测分析其临床诊断效能。结果：将含有梯度数量的曲霉菌孢子的样本提取总RNA后进行NASBA-ELISA检测，结果表明光密度值与霉菌孢子量对数存在线性相关关系（y=0.278x+0.119，R2=0.887），该方法的灵敏度可达1个孢子；对照菌株（大肠埃希菌、金黄色葡萄球菌、铜绿假单胞菌、白色念珠菌和新型隐球菌）和曲霉菌提取RNA后进行检测，结果仅曲霉菌的吸光度（absorbance，A）值大于设定的阈值（6个随机阴性标本的平均A值+3倍标准差）呈阳性；对82例临床标本进行NASBA-ELISA检测，其敏感度和特异度分别为80.6%、80.0%，受试者工作特征曲线曲线下面积为0.759（95%CI=0.644~0.874）。结论：NASBA-ELISA方法检测曲霉菌具有敏感性高、特异性强、操作简便的特点，适合常规实验室开展，为侵袭性曲霉病的早期诊断提供了一条新方法。

Abstract:

Objective：To develop a nucleic acid sequence-based amplification coupled ELISA method for detection of invasive as－pergillosis（IA）. Methods：In this experiment，the nucleic acid sequence-based amplification was combined to the high sensitive sys－tem of digoxingenin. The specific 18sRNA of Aspergillus was amplified by the isothermal digoxigenin（DIG）-labeling NASBA process，then the DIG-labeled NASBA amplicons were immobilized on streptavidin-coated microtiter plate by hybridized with a specific bi－otinylated DNA probe. The hybrids were colorimetrically detected by the addition of an anti-DIG antibodies linked to AIP and sub－strate（disodium 4-nitrophenyl phosphate）. The specificity，sensibility and the clinical diagnostic efficiency of this method were eval－uated by detecting the total RNA extracted from the ten-fold serially titrated Aspergillus spores，the RNA extracted from the compar－isons and 82 blood samples（32 positive cases，50 negative cases）. Results：The total RNA extracted from the ten-fold serially titrated Aspergillus spores was detected by NASBA-ELISA. The optical density value was linearly correlated with the logarithm of the spore amount（y=0.278x+0.119，R2=0.887），and this method could detect as little as 1 CFU aspergillums spore. The RNA extracted from the comparisons（E.coli，S.aureus，P.Aeruginosa，C.albicans，C.neoformans） and Aspergillus were detected by NASBA-ELISA. The results showed that NASBA-ELISA was highly specific for Aspergillus detection and did not cross-react with the non-target fungi or bacte－ria. The performance of NASBA-ELISA system was evaluated by detecting blood samples of 82 patients at high risk for IA. The sensitivity and specificity were 80.6% and 80.0%. The NASBA-ELISA A values were assessed by ROC analysis，area under the curve=0.755（95%CI=0.645 to 0.865）. Conclusion：The NASBA-ELISA method can detect Aspergillus sensitively and specifi－cally，so it provides a new way for the molecular epidemiologic investigation and early diagnosis of IA.