包装饮用水中痕量铜绿假单胞菌核酸试纸条快速、可视化检测分析

Rapid Visual Detection of Trace Pseudomonas aeruginosa in Packaged Drinking Water Using Nucleic Acid Test Strips

目的:建立基于胶体金标记的侧向层析免疫试纸快速检测新技术检测包装饮用水中铜绿假单胞菌的方法。方法:利用喷膜仪以0.5μL/cm的喷速将1 mg/mL的羊抗鼠抗体、1 mg/mL的链霉亲和素喷到硝酸纤维素膜上,再通过静电吸附的方式将异硫氰酸荧光素抗体偶联到胶体金表面,制备成偶联物滴加到金标垫上。将扩增产物滴加到组装完整的侧向层析免疫试纸条上,根据质控、检测线位置的显线情况进行判定。结果:该方法能够对包装饮用水中的铜绿假单胞菌进行特异性识别鉴定,3 min便可获得检测结果,检出限为1 CFU/250 mL,较琼脂糖凝胶电泳结果更灵敏。结论:本研究建立的对包装饮用水中铜绿假单胞菌的侧向免疫试纸方法具有灵敏度高、特异性强、简单便携等技术优势,可用于实际样本的检测。

Objective:To establish a new method for the rapid detection of Pseudomonas aeruginosa in packaged drinking water using colloidal gold-based lateral flow immunochromatographic strips.Methods:1 mg/mL sheep anti-mouse antibody and 1 mg/mL streptavidin(SAV) were sprayed onto a nitrocellulose(NC) membrane by using a membrane sprayer at a rate of 0.5 μL/cm,and then the antibody labeled with fluorescein isothiocyanate(FITC) was conjugated to the surface of colloidal gold by electrostatic adsorption,and the resulting conjugate was added dropwise to the gold standard pad.The amplification products were added dropwise to the fully assembled lateral flow immunochromatographic strip,and the results were judged based on the positions of the control(C) and test(T) lines generated.Results:The method was able to specifically identify P.aeruginosa in packaged drinking water in 3 min with a detection limit of 1 CFU/250 mL,which was more sensitive than agarose gel electrophoresis.Conclusion:The lateral flow immunoassay method was characterized by high sensitivity,high specificity,simplicity and portability,and was suitable for real samples.