基于不对称聚合酶链式反应的多靶标纸基核酸芯片用于水源性致病菌的同步检测OA

Waterborne pathogenic bacteria can be transmitted via contaminated water bodies,causing a variety of illnesses including diarrhea and systemic sepsis,and posing a serious threat to human health.Therefore,the development of rapid and sensitive detection methods for these pathogenic bacteria is of great importance for the timely identification of pollution sources and the control of disease transmission.In this study,by coupling asymmetric polymerase chain reaction(PCR)with colloidal gold-based nucleic acid lateral flow chromatography technology,a highly sensitive novel method was developed for simultaneous detection of four kinds of common waterborne pathogenic bacteria using a multi-channel paper-based strip chip.By adopting an asymmetric design of primer concentrations and optimizing key parameters including primer ratio and cycling procedures,this method enabled the generation of abundant target gene-containing single-stranded DNA(ssDNA)products during amplification,which significantly improved the hybridization efficiency between the ssDNA products and the immobilized probes on the strip,thereby enhancing the detection sensitivity.On this basis,specific capture probes corresponding to the target sequences were coated onto a nitrocellulose(NC)membrane to serve as test lines(T lines),and a control line(C line)was prepared using a streptavidin-biotinylated DNA conjugate.The multi-channel paper-based nucleic acid chip was then fabricated by assembling the modified NC membrane with a gold conjugate pad,sample pad,absorbent pad,and polyvinyl chloride(PVC)backing plate.The results demonstrated that the developed chip exhibited strong specificity,high sensitivity,and visualization-enabled readout,with limits of detection of 17.3,13.2,16.6 and 19.7 ng/μL for Vibrio parahaemolyticus,Staphylococcus aureus,Salmonella enterica,and Escherichia coli,respectively.In detection of real tap water samples,this method exhibited excellent anti-matrix interference ability,with spiked recoveries ranging from 92.5%to 106.8%,which held promising application prospects for on-site rapid screening of multiple bacterial contamination in water bodies.