肠毒素大肠杆菌K88可视化快速检测方法的建立

Establishment of a Visual and Rapid Detection Method for Enterotoxigenic Escherichia coli K88

本研究通过肠毒素大肠杆菌（E.coli）K88菌毛蛋白的适配体识别,结合纳米金标记和银增强信号放大技术,建立了一种快速、灵敏、特异的E.coli K88可视化快速检测方法。该检测方法是将能与E.coli K88特异性结合的生物素化的适配体1（aptamer 1）,与目标菌E.coli K88以及纳米金-巯基化适配体2轭合物（aptamer 2-Au NPs）在一定条件下孵育,形成三明治式的aptamer 1-E.coli K88-aptamer 2-Au NPs复合物,随后通过生物素与亲和素的结合将复合物固定到修饰了链霉亲和素的微孔板上,最后运用银增强显色将反应信号放大。通过对检测方法条件的优化,本方法可特异、定量地检测E.coli K88,在1.0×10^1-1.0×10^5 cfu/孔目标菌范围内,其定量拟合线性曲线决定系数R^2可达0.9903,且检测灵敏度达10 cfu/孔,而检测其它非目标菌株均为阴性。本方法为E.coli K88在临床样品中的可视化检测奠定了基础。

A method that could rapidly, sensitively, specifically, and visually detect E. coli K88 was established based on the aptamer recognition of the E. coli K88 fimbriae protein, combined with gold nanoparticles （AuNPs） labeling and silver enhancement signal amplification. Biotinylated aptamer 1 that could specifically bind to E. coli K88 was incubated with target bacterium E. coli K88 and the AuNPs-aptamer 2 conjugates, to form a sandwich-type aptamer 1-E. coli K88-aptamer 2-AuNPs complex. Then, the sandwich-type complex was immobilized onto the surface of a 96-microwell plate modified with streptavidin through the binding of biotin and streptavidin. Finally, the responding signal was amplified using silver enhancement. After the optimization of the detection conditions, this method could detect E. coli K88 specifically and quantitatively. When the E. coli K88 concentration was in the range of 1.0×10^1-1.0×10^5 cfu/well, the coefficient of determination （R^2） for quantitative linear curve fitting was 0.9903, the detection sensitivity could reach 10 cfu/well, and negative results were obtained in the detection of other non-target bacterial strains. This paper provides a basis for the visual detection for E. coli K88 in clinical samples.