纳米金标记-逐步银染法快速检测金黄色葡萄球菌

Nanogold Labeling-stepwise Silver Staining Method for Rapid Detection of Staphylococcus aureus

将纳米金探针应用于目的核酸的检测,具有与PCR相当的灵敏度和特异性.本研究建立了一种可以在微孔板上快速检测金黄色葡萄球菌的纳米金标记-逐步银染法.该方法利用已包被链霉亲和素的微孔板,将PCR扩增的金黄色葡萄球菌nuc基因与生物素探针、纳米金探针形成的三明治杂交结构锚定其上,然后在低温下逐步银染显色,通过酶标仪检测放大的银染信号.这种纳米金标记-逐步银染法可以在显著降低非特异性背景信号的同时放大银染信号,检测金黄色葡萄球菌nuc基因的灵敏度为1 pmol/L,比常温一步银染法的灵敏度提高约102倍.51例临床标本的检测结果与PCR法一致,与培养生化鉴定法的检测结果之间无显著性差异(P>0.05).综上所述,本研究成功构建了金黄色葡萄球菌的纳米金标记-逐步银染法,在病原微生物的快速检测领域表现出广阔的发展潜力.

The methods of gold nanoparticle probes for the detection of nucleic acid targets have advantages including PCR-like sensitivity and selectivity for target sequences. A new nanogold labelingstepwise silver staining method on microplate was described for rapid detection of Staphylococcus aureus. The method involves forming the sandwich structures by the hybridization of PCR amplification products of S. aureus 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, which can reduce non-specific background signal and meanwhile amplify silver staining signal. The detection limitation of S. aureus hue gene was 1 pmol/L. The sensitivity of this method exceeds that of one-step silver staining at room temperature by two orders of magnitude. The results of nanogold labeling-stepwise silver staining method were consistent with PCR for detecting S. aureus in fifty-one clinical specimens, and there was no significant difference between this method and clinical microbiological procedures （P 〉 0.05 ）. In conclusion, we successfully established a nanogold labeling-stepwise silver staining method on microplate for detection of S. aureus. It would have a broad developing prospect in the rapid detection of pathogenic microorganisms.