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目的对基于酪胺信号放大和金标银染技术的致病菌可视化生物芯片检测方法进行评价。方法以伤寒沙门氏菌和痢疾志贺菌作为检测对象,建立生物芯片的TSA-金标银染高灵敏度可视化检测技术,优化试剂浓度、反应温度、温育及银染时间等检测条件,比较了TSA-金标银染与单独金标银染、TSA-金标银染与TSA-荧光的检测灵敏度。并对进口水产品样品进行了伤寒沙门氏菌和痢疾志贺菌的应用检测。结果检测条件优化结果为:Streptavidin-HRP稀释比例为1:1 500,Biotin-Tyramine稀释比例为1:200+0.5%H2O2,Streptavidin-Nanogold稀释比例为1:100;温育温度37℃,温育时间20 min,银染显色时间8～10 min。TSA-金标银染比单独金标银染的检测灵敏度提高10～100倍,与TSA-荧光的检测灵敏度相同,达到103CFU/ml。检测进出口水产品样品伤寒沙门氏菌和痢疾志贺菌结果与SN标准方法检测结果一致。结论基于酪胺信号放大和金标银染技术的致病菌可视化生物芯片检测方法,为致病菌低成本快速检测提供了新思路。
Objective To evaluate the visual biochip detection method based on tyramine signal amplification and gold standard silver staining. Methods Salmonella typhimurium and Shigella dysenteriae were used as detection objects to establish the TSA-gold-labeled high-sensitivity visualization detection technology of biochips. The detection conditions of reagent concentration, reaction temperature, incubation and silver staining time were optimized. The TSA- Gold silver and silver gold staining alone, TSA-gold silver staining and TSA-fluorescence detection sensitivity. The samples of imported aquatic products were tested for the application of Salmonella typhi and Shigella dysenteriae. Results The optimized conditions were as follows: 1: 1500 Streptavidin-HRP, 1: 200 + 0.5% H2O2 for Biotin-Tyramine, 1: 100 for Streptavidin-Nanogold, 37 ℃ for incubation Time 20 min, silver staining time 8 ~ 10 min. The detection sensitivity of TSA-gold silver is 10 to 100 times higher than that of silver gold alone, which is the same as that of TSA-fluorescence and reaches 103CFU / ml. Detection of import and export of aquatic samples Salmonella typhi and dysentery Shigella results consistent with the SN standard method test results. Conclusion The visualization biochip detection method based on tyramine signal amplification and gold silver staining technology provides a new idea for rapid detection of pathogenic bacteria at low cost.