Nanogold Labeling-stepwise Silver Staining Method For Rapid Detection Of Staphylococcus Aureus

Objective:To develop a nanogold labeling-stepwise silver staining method on microplate for rapid detection of Staphylococcus aureus, and apply the method to rapid detection of various clinical specimens.Method:Gold nanoparticles are synthesized by using HEPES as a reductant. Then nanogold-labeled probes are prepared by these particles coupled with sulfhydryl modified oligonucleotide probes. The method involves forming the sandwich structures by the hybridization of PCR amplification products of Staphylococcus aureus specific nuc gene with biotin-labeled probes and nanogold-labeled probes. When the sandwich structures were anchored on the streptavidin-coated clear plate, the hybridization signals detected by a microplate reader were amplified by stepwise silver staining under low temperature. The detection limitation, specificity and reproducibility of this method were evaluated. Fifty-one clinical specimens were detected by this method, and the results were compared with that of PCR and clinical microbiological procedures.Results:The result shows that gold nanoparticles with low reaggregation and average size of about12nm are synthesized. The wavelength at the maximum of the absorption band red-shifts about5nm in nanogold-labeled probes. The optimized condition of nanogold labeling-stepwise silver staining method on microplate was as follows: the temperature and staining time of stepwise silver staining was4℃,5min4; the concentration of biotin-labeled probes was100nmo/L; the concentration of nanogold-labeled probes was20nmo/L. This method can reduce non-specific background signal and meanwhile amplify silver staining signal. The detection limitation of Staphylococcus aureus nuc gene was1pmol/L, which exceeds that of one-step silver staining at room temperature by two orders of magnitude. All the other bacterial strains tested were negative. The intra-and inter-assay coefficient of variation of nanogold labeling-stepwise silver staining method for600pmol/L and6nmol/L target sequence were less than10%. The results of nanogold labeling-stepwise silver staining method were consistent with PCR for detecting Staphylococcus aureus in fifty-one clinical specimens, and there was no significant difference between this method and clinical microbiological procedures (P>0.05)Conclusion:We successfully established a nanogold labeling-stepwise silver staining method on microplate for detection of Staphylococcus aureus, which is simple and fast with high sensitivity and strong specificity. And the results can be detected by microplate reader without special instrument. It would have a broad developing prospect in the rapid detection of pathogenic microorganisms.