Detection Of Nucleic Sequence-based Amplification Products By Gold Nanoprobe-based Solution Hybridization For The Diagnosis Of Invasive Aspergillosis

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）.Then the GM test was also evaluated to find out the optimal clinical laboratory diagnostic strategy of the infections.Methods:The oligonucleotide probes were designed for 18S rRNA conserved sequence of Aspergillus and were decorated with sulfhydryl at the 5’end.Then the gold-nanoprobes were prepared by assembling the sulfhydryl modified oligonucleotide probes onto the surface of the 60nm diameter gold nanoparticles.The 18s rRNA of Aspergillus and non-Aspergillus were amplified by NASBA,the amplified products were hybridized with gold nanoprobes.The specificity was investigated by observing the color changes of the reaction system or its agglutination on the reverse-phase chromatography plate.The definitions of invasive fungal infection according to the EORTC/MSG updated criterion are classified into“proven”,“probable”and“possible”.Based on this criterion,blood samples from 106 patients（including 14 proven cases,32 probable cases,60 without IA cases）were detected by the established NASBA-gold nanoprobe method,and the obtained results were compared with that of galactomannan（GM）test to evaluate its accuracy.Prospective comparison of the two methods was performed to find out the optimal clinical laboratory diagnostic strategy of IA.Results:The gold nanoprobes only hybridized with Aspergillus NSABA products with the color changes from wine red to grey or black agglutination particles observed on the reverse-phase chromatography plate,while other non-Aspergillus strains are negative.The sensitivity,specificity and the area under the ROC curve(AUC^ROC)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(AUC^ROC)of GM test were 56.52%（26/46）,83.33%（50/60）and 0.723,respectively.Combination of the NASBA-gold nanoprobe method and GM-ELISA achieved perfect specificity（100%）and perfect positive predictive value（100%）.The best sensitivity（89.13%）and nagative predictive value（88.89%）were obtained by testing with both NASBA-gold nanoprobe method and GM-ELISA in parallel.Conclusion:The established GNBSH method to detect Aspergillus NASBA products has the characteristics of high sensitivity,high specificity and simple operation,which may be used to detect the infection of Aspergillus by clinical laboratories.Combination assay provides a new way for the molecular epidemiologic investigation and early diagnosis of IA.