基于酪胺信号放大和金标银染的致病菌生物芯片检测方法的灵敏度评价研究

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。

Study on sensitive evaluation of bio-chip detection for pathogenic bacteria based on tyramine signal amplification coupled with gold label silver stain

Objective To evaluation a rapid, sensitive bio-chip visual detection of two kinds of pathogenic bacteria in aquatic products based on tyramine signal amplification and gold label silver stain. Methods Salmonella typhi and shigella spp were selected as the targets in this study, a sensitive and visual detection method that based on TSA coupled with GLSS was developed. The procedure was set and detection parameters included dilution ratio of the reagents, incubation temperature, and silver stain time et al., was systematically optimized. Detection sensitivity among TSA-GLSS, GLSS and TSA-Cy3 was compared. The method was applied to samples collected from aquatic products for entry and exit. Results The optimized parameters were listed as follows： streptavidin-HRP dilution of 1：1 500, biotin-tyraminc dilution of 1：200 and 0.5% H：O2 was added, streptavidin-Nanogold dilution of 1：100, incubation time of 20 min, and silver stain time of 8~10 min. The sensitivity of TSA-GLSS was enhanced 10-100 fold compared with that of GLSS, and was comparable with that of TSA-Cy3. Detection limit of TSA-GLSS and TSA-Cy3 both reached 103 CFU/ml. The detection results of samples collected from aquatic products for entry and exit with the method were consistent with those of SN reference methods. Conclusion It is proved that the bio-chip visual detection of pathogenic bacteria in aquatic products based on tyramine signal amplification and gold label silver stain is a rapid, sensitive.