酪胺信号放大-量子点标记银染增强的基因芯片可视化检测方法的建立

目的:建立一种酪胺信号放大-量子点标记银染增强的基因芯片可视化检测方法,提高基因芯片检测的灵敏度。方法:待测靶基因与固定在玻片上的探针杂交,依次加入链霉亲和素标记的辣根过氧化物酶、生物素标记的酪胺及链霉亲和素标记的量子点,37℃孵育,然后加入银增强试剂显色,最后用可视化生物芯片扫描仪扫描并记录结果;以牛布鲁菌210105株为检测对象,以酪胺信号放大-荧光素Cy3(TSA-Cy3)检测法为对照方法,测定酪胺信号放大-量子点标记银染增强(TSA-QDS)检测法的灵敏度。结果:确定了基因芯片量子点标记银染增强可视化检测方法的检测流程,优化了检测条件,并考察了检测灵敏度。优化的检测条件为:酪胺-生物素稀释比例为1∶4000,链酶亲和素标记的量子点稀释比例为1∶50,37℃孵育时间为25~30 min,银染增强时间为6~7 min。检测牛布鲁菌的灵敏度为103CFU/mL。结论:该方法实现了基因芯片高灵敏度可视化检测,其灵敏度与荧光法相当,并且有可视化的优势。

Development of Visual Detection Based on TSA-Quantum Dots Cou pled with Silver Enhancement for DNA Microarray

Objective: To establish a novel method for DNA microarray of visual detection based on tyramine signal amplification and quantum dots(QD) coupled with silver enhancement.Methods: Each zone of the slide was hybrided with the target DNA and loaded with the dilution of streptavidin-HRP,biotin-tyramine,streptavi din-QDs in turn,and incubation was necessary at 37℃ after each dilution loaded.The images results were ob tained after silver staining and a visible light scanner detecting.Meanwhile,the procedure was systematically opti mized.Detection sensitivity of TSA-QDS(TSA-quantum dots-silver enhancement) was measured when Brucella abortus strain 210105 was used as target and TSA-Cy3 as positive control method.Results: The procedure was de termined and detection parameters including dilution ratio of the regents,incubation temperature,and silver stain time as well.The optimized biotin-tyramine dilution was 1∶4000,streptavidin-QDs 1∶50,the glass slides were in cubated at 37℃ for 20~30 min and stain time 6~7 min.Detection limit of the method reached 103 CFU/mL for B.abortus.Conclusion: A new method for DNA microarray of visual detection was developed,the sensitivity of which was comparable to that of TSA-Cy3,more important is that the visual detection was realized.