一种基于适配体识别和酪胺信号放大的鼠伤寒沙门菌检测方法

A detection method for Salmonella typhimurium based on aptamer recognition and tyramine signal amplification

目的针对鼠伤寒沙门菌全菌细胞,建立了一种基于适配体识别和酪胺信号放大技术的鼠伤寒沙门菌检测方法。方法利用适配体的特异性识别作用将鼠伤寒沙门菌进行捕获并且通过亲和素和生物素的连接作用将其固定到酶标板的底部,然后利用酪胺信号放大技术对信号进行放大,再依次加入辣根过氧化物酶(HRP)标记的链霉亲和素、3,3′,5,5′-四甲基联苯胺(TMB),最后加入终止液,在室温下放置20 min后,产生的黄色产物就可以利用酶标仪进行检测。结果在最优的检测条件下,鼠伤寒沙门菌浓度在10 cfu/ml~10;cfu/ml内,A;的值与平板计数法得到的菌落个数的对数值具有良好的线性关系(r=0.9988),检测限为9 cfu/ml。结论本方法能够能够实现对样品中鼠伤寒沙门菌的快速、灵敏的检测。

Objective A detection method for the detection ofSalmonella typhimuriumbased on aptamer recognition coupled to tyramine signal amplification technology has been developed with the target of the detection of the wholeSalmonella typhimurium cell.Methods The biotinylated aptamer specific forSalmonella typhimuriumwas immobilized on the surface of the wells of a microplate via biotin-avidin binding.Then,the target bacteria(Salmonella typhimurium),the biotinylated-aptamer-streptavidin-HRP conjugates,biotinylated tyramine,hydrogen peroxide and streptavidin-HRP were successively placed in the wells of the microplate.After adding TMB reagent and stop solution,the intensity of the yellow reaction product can be measured with a plate reader after 20 min at room temperature.Results Under the optimal detection conditions,the concentration ofSalmonella typhimuriumranged from 10 cfu/ml to 10~7cfu/ml,the value ofA;had a good linear relationship with the logarithm of the colony number obtained by plate counting method(r=0.9988),and the detection limit was 9 cfu/ml.Conclusion This developed method offers the potential for rapid,sensitive detection ofSalmonella typhimuriumin samples.