Nanogold Labeling And Silver Staining Method For Rapid Detection Of Mycobacterium Tuberculosis In Sputum Samples

Objective:To develop a nanogold labeling silver staining method on microplate for rapid detection of Mycobacterium tuberculosis, and apply the method to rapid detection of suspicious clinical sputum specimens.Methods:The nanogold-labeled probes are prepared by gold nanoparticles coupled with sulfhydryl modified oligonucleotides. The repetitive insertion sequence IS6110was used as target gene, then two kinds of probes, which were labeled respectively with biotins and gold nanoparticles, were respectively complementary to one half of the target gene to form a sandwich structure. The hybridization products of target gene with these two kinds of probes were immobilized to microplates via streptavidin-biotin system, and the signals of gold nanoparticles were subsequently amplified by silver staining solution, and recorded with colorimetric absorbance. Mycobacterium tuberculosis in90clinical sputum samples were detected simultaneously by this mothed, clinical microbiological procedures and PCR.Results:The wavelength at the maximum of the absorption band red-shifts about4nm in nanogold-labeled probes. The optimized condition of nanogold labeling silver staining method on microplate was as follows:the staining time of silver staining enhancement was8min; the concentration of biotin-labeled probes was100nmo/L; the concentration of nanogold-labeled probes was25nmo/L. This method can reduce non-specific background signal and meanwhile amplify silver staining signal. This sandwich colorimetric assay can detect as few as1pmol/L each tube, and all the other bacterial strains tested were negative. The intra-and inter-assay coefficient of variation of nanogold labeling-stepwise silver staining method for1nmol/L,100pmol/L and1pmol/L target sequence were less than7%. The results of nanogold labeling silver staining method were consistent with PCR for detecting Mycobacterium tuberculosis in ninety clinical sputum samples, and there was no significant difference between this method and clinical microbiological procedures(P>0.05).Conclusion:In this study, we successfully prepared the nanogold-labeled probes by sulfhydryl modified oligonucleotides coupled with gold nanoparticles. And successfully constructed nanogold labeling and silver staining method, which can rapidly detect Mycobacterium tuberculosis in clinical sputum samples, could be an accurate, convenient and economical assay for clinical application.