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

    陆琳，车志军，孙福军，杨秀娟，刘国传

    北京出入境检验检疫局，北京 100026

    摘要：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20min，银染显色时间8~10min。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。

    关键词：致病菌；酪胺信号放大；金标银染；生物芯片

    中图分类号：R155.5　文献标识码：B

    Study on sensitive evaluation of bio-chipdetection for pathogenic bacteria

    based on tyramine signal amplificationcoupled with gold label silver stain

    LU Lin, CHE Zhi-jun, SUN Fu-jun, YANGXiu-juan, LIU Guo-chuan

    Beijing Entry-exit Inspection and QuarantineBureau, Beijing 100026,China

    Abstract:   Objective  To evaluation a rapid, sensitive bio-chip visual detection of twokinds of pathogenic bacteria in aquatic products based on tyraminesignal amplification and gold label silver stain.  Methods   Salmonella typhi and shigella spp were selectedas the targets in this study, a sensitive and visual detectionmethod that based on TSA coupled with GLSS was developed. Theprocedure was set and detection parameters included dilution ratioof the reagents, incubation temperature, and silver stain time etal., was systematically optimized. Detection sensitivity amongTSA-GLSS, GLSS and TSA-Cy3 was compared. The method was applied tosamples collected from aquatic products for entry andexit.   Results   The optimized parameters werelisted as follows: streptavidin-HRP dilution of 1:1 500,biotin-tyramine dilution of 1:200 and 0.5% H2O2  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, andwas comparable with that of TSA-Cy3. Detection limit of TSA-GLSSand TSA-Cy3 both reached 103 CFU/ml. The detection results ofsamples collected from aquatic products for entry and exit with themethod  were consistent with those of SN referencemethods.  Conclusion   It is proved that  thebio-chip visual detection of pathogenic bacteria in aquaticproducts based on tyramine signal amplification and gold labelsilver stain is a rapid, sensitive.

    Key words:   Pathogenic Bacteria;Tyramine signal amplification; Gold label silver stain;Bio-chip

    《中国国境卫生检疫杂志》2012年第6期