纳米金探针液相杂交法检测核酸序列依赖扩增产物诊断侵袭性曲霉病

Detection of nucleic acid sequence-based amplification products by gold nanoprobe-based solution hybridization for the diagnosis of invasive aspergillosis

目的建立纳米金探针液相杂交法检测核酸序列依赖扩增(nucleic acid sequence-based amplification,NASBA)产物,用于快速诊断侵袭性曲霉病(IA)。方法采用NASBA扩增曲霉菌属特异性18S rRNA保守序列,扩增产物与5'端经巯基化俢饰的特异性纳米金探针进行液相杂交,用该法检测106例临床血液标本(14例确诊,32例拟诊,60例未感染IA),并与GM试验结果进行比较以评价该方法的准确性。结果纳米金探针只与曲霉菌属菌株的NASBA产物出现阳性杂交反应,而其他非曲霉菌均为阴性。NASBA-纳米金液相杂交法检测106例临床标本的敏感性和特异性分别为82.61%(38/46)、81.67%(49/60),ROC曲线下面积(AUC^(ROC))为0.890。GM试验检测的敏感性和特异性分别为56.52%(26/46)和83.33%(50/60),AUCROC为0.723。结论 NASBA-纳米金液相杂交技术检测曲霉菌感染具有敏感性高、特异性强、操作简单的特点,适合实验室常规开展。

Objective To establish a method of gold nanoprobe-based solution hybridization（ GNBSH） to detect nucleic acid sequence-based amplification（ NASBA） products for the rapid diagnosis of invasive aspergillosis（ IA）. Methods The Aspergillus specific 18 S rRNA was amplified by NASBA and then the amplified products were hybridized with the gold nanoprobes which were modified with thiol compounds at the 5＇ end. Serum samples from 106 patients,including 14 with a definite IA,32 with suspected IA and 60 without IA,were detected by the established method,and the obtained results were compared with that of galactomannan（ GM） test to evaluate its accuracy. Results The gold nanoprobes only hybridized with Aspergillus NASBA products but not other non-Aspergillus strains. The sensitivity,specificity and the area under the ROC curve（ AUCROC） of the established GNBSH method for detecting 106 clinical samples were 82. 61%（ 38/46）,81. 67%（ 49/60） and 0. 890,respectively. The sensitivity,specificity and AUCROCof GM test were 56. 52%（ 26/46）,83. 33%（ 50/60） and 0. 723,respectively. Conclusion The established GNBSH method to detect Aspergillus NASBA products has high sensitivity and specificity and simple operation,which may be used to detect the infection of Aspergillus by clinical laboratories.