The Selection And Application Of Aptamers Targeted To Foodbornpathogenic Bacteria

Aptamers are short, single-stranded RNA or DNA molecules that can bind with highaffinity and specificity to a target molecules. They are generated in an in vitro systematiciterative process, which is known as SELEX (systematic evolution of ligands by exponentialenrichment). Due to its high affinity and specificity, aptamers are widely used in basicresearch, proteomics research, diagnostic and therapeutic. However, there is few research onfood safety. In this study, high-affinity ssDNA aptamers binding to foodborn pathogenicbacteria were screened. As a recognition element, it was used to combine magneticnanoparticles separation and concentration, upconversion nanoparticals, flow cytometry andlabel-free aptasensor to establish a series of novel, sensitive and rapid detection methods forfoodborn pathogenic bacteria, which provide a new means for enriching and improvingtechnology of detection in food safety.Firstly, a whole-bacteria Systemic Evolution of Ligands by Exponential Enrichment(SELEX) method was applied to identify DNA aptamers demonstrating specific binding toVibrio parahaemolyticus. After nine rounds of selection, a highly enriched oligonucleotidepool was sequenced. The binding affinity and specificity assay were analysed by flowcytometry. The sequence, which presented high affinity and specificity with the target, wasthe aptamer of Vibrio parahaemolyticus. Based on the same principle and method, ahigh-affinity ssDNAaptamer binding to Salmonella typhimurium was obtained. Moreover, theconsensus sequence of the two aptamers were also analysed by removing base one by one.Secondly, a sensitive luminescent bioassay for the simultaneous detection of Salmonellatyphimurium and Staphylococcus aureus was developed using aptamer-conjugated magneticnanoparticles (MNPs) for both recognition and concentration elements and usingupconversion nanoparticles (UCNPs) as highly sensitive dual-color labels. The correlationbetween the concentration of Salmonella typhimurium and luminescent signal was found to belinear within the range of101~105cfu/mL (y=169.66X-35.2, R~2=0.9964), with the limits ofdetection5cfu/mL, and101~105cfu/mL (y=118.5X+15.6, R~2=0.9936) for Staphylococcusaureu, with the limits of detection8cfu/mL. The bioassay was successfully applied to detectSalmonella typhimurium and Staphylococcus aureus in real water samples.Thirdly, a dual functional platform was demonstrated for the simultaneous detection oftwo pathogenic bacteria using quantum dots as fluorescence markers coupled with aptamersas the molecular recognition element by flow cytometry. The results were compared with theexperimental results from plate-counting methods (for Vibrio parahaemolyticus P<0.0001,R~2=0.9993; for Salmonella typhimurium P<0.0001, R~2=0.9995). The bioassay wassuccessfully applied to detect Vibrio parahaemolyticus and Salmonella typhimurium in realshrimp samples. This is the first time for the application of aptamer-conjugated QDs in flowcytometry for bacteria determination.Fourthly, a fluorescence detection of foodbore pathogenic bacteria using a label-freeaptasensor was researched in this section. The fluorescence of AccuBlue reagent itself is veryweak, but it becomes highly fluorescent upon binding to dsDNA, while no significant fluorescence change can be observed with its binding to ssDNA and impermeable to cellmembranes. This unique phenomenon of AccuBlue has laid the foundation for thedevelopment of the label-free aptasensor herein. Two strategy of “signal on” and “signal off”was proposed to detect Vibrio parahaemolyticus and Salmonella typhimurium, respectively.The correlation between the concentration of Vibrio parahaemolyticus and luminescent signalwas found to be linear within the range of50~106cfu/mL (y=197.23x+15.688, R~2=0.9956),and the limits of detection was35cfu/mL. The correlation between the concentration ofSalmonella typhimurium and luminescent signal was found to be linear within the range of50~106cfu/mL (y=183.72x+30.79, R~2=0.9957), and the limits of detection was25cfu/mL.The proposed methods had the ability to effectively detect the real shimp sample and chickensample.Fifthly, the binding mechanism of aptamer and target molecules was preliminary studiedby detecting the affinity between aptamers and surface molecules of the bacteria. The bacteriaouter membrane proteins (OMPs) and lipopolysaccharide (LPS) was analysed to be the mostlikely surface molecules to aptamer. The OMPs and LPS were isolated and incubated withtheir corresponding aptamers, and the binding affinity was analysed using fluorescencepolarization. The results indicated that the affinity of Vibrio parahaemolyticus, or Salmonellatyphimurium aptamer and OMPs is higher than that of LPS. That is to say aptamers, generatedby whole-bacteria SELEX, bind to OMPs with specificity. The results had a positive value forclarifying the binding mechanism of aptamer and target molecules.In conclusion, aptamers targeted to foodborn pathogenic bacteria were selected as anovel recognition element. Benefit from its high affinity and specificity to targets and simplemodification, a series of novel, sensitive and rapid detection methods were established for oneor two bacteria determination combining with UCNPs, QDs, flow cytometry. That provides atechnical support for developing detection methods and relative products based on aptamersfor pathogenic bacteria.