Detection Of Food-borne Pathogenic Bacteria With CdSe/ZnS Quantum Dots Modified By Streptavidin

Quantum dots is a new semiconductor nanocrystal material. As a fluorescent probe,it has many unique excellent optical characteristics compared with traditional organicdyes. Firstly, as QDs have broad effective excitation spectra and the narrow andsymmetrical emission of QDs. simultaneous excitation of multiple QDs can beaccomplished easily with a single excitation ligh. Secondly,the fluorescence strength ofQDs do not decrease when exposed to light for long periods of time (that is, QDs arephotostable). Finally, although QDs with different sizes have different emissionwavelengths (different colors), they have similar physical properties (such asdimension and polarity) and biological conjugation ability (namely, that caneffectively conjugate to biomolecules). In recent years, quantum dots have been widelyused in genetic analysis, immunoassay analysis and microbiological testing. This thesismainly focues on developing a new method that employs the techniques ofoligonucleotide microarray combined with quantum dots as fluorescent labels andexploring the effects of CdSe/ZnS quantum dots modified by streptavidin onPolymerase Chain ReactionwereThe main contents and results of this thesis are summarized as follows:1．Detection of foodborne pathogenic bacteria by gene chip with CdSe/ZnS quantumdots modified by streptavidin as fluorescent labels.With the feature of high-speed, high-efficiency and high-throughput, gene chiptechnology has a promising prospect in the rapid diagnosis microorganisms. Currentlygene chip technology has been widely applied in DNA detection and genotyping,however, thay have demerits in low sensitivity and stability. To date, the labelmolecules use in DNA microarrays is fluorescent organic dyes which suffer fromseveral limitation (such as low signal intensities, rapid photobleaching, a narrowexcitation). All this disadvantages can be avoided by the use of quantum dots. In thispaper, we develop a new method which employs the technique of oligonucleotidemicroarray combined with quantum dots as fluorescent labels and we used this methodto simultaneously detect multiple food-borne bacterial pathogens. Oligonucleotide probes targeted to16s rDAN were synthesized to create anoligonucleotide microarray in this study. The PCR products labeled by biotin weresubsequently hybridized with the oligonucleotide microarray. After incubation withCdSe/ZnS quantum dots coated with streptavidin, fluorescent signals were reflected bya PerkinElmer Gx Microarray Scanner.It is found that a wide variety of food-borne pathogenic bacteria including Vibrioparahaemolyticus, Vibrio fluvialis, Yersinia enterocolitica, Proteus spp.,Staphylococcus aureus, Enterococcus faecalis, Campylobacter jejuni, Streptococcushemolytic-β, Listeria monocytogenes, can be clearly discriminated by this means whilestrains belonging to Escherichia coli, Shigella spp., Salmonella spp., are differentiatedas one class of pathogenic bacteria. This method has a high specificity.One of the key parameters in detection methods is sensitivity. In order to improvethe sensitivity of gene chip we can choice good label molecules and signal amplicationsystem. The good label molecules should have such features as safe, sensitive,non-radioactive, high intensity of fluorescence, stability of the chemical properties.Fortunately, the quantum dots have all of the above characteristics. In this paper, wealso introduces the biotin-streptavidin system which has a signal amplification effect,combined with quantum dots as fluorescent labels to enhance the sensitivity of the DNAmicroarray. The results show that the sensitivity of this method is10cfu/mL of pureculture. The repeatability of this method is also good.In order to assess the ability of application in the food amples,78strains ofpathogenic bacteria isolated from food samples were processed and detected by thismethod maps. All78strains were distinguished by their hybridization maps. Thecomprehensive identification results by classical methods are regarded as the finalstandards. All the hybridization assay results were consistent with those of theconventional methods and the consistency was100%.Pathogenic bacteria can be quickly detected and accurately identified within7to8hby use of this method. Moreover, the strategy and approach may be easily expanded toany other bacteria, thus it establishes a revolutionary basis for detecting pathogenicbacteria by oligonucleotide microarray with QDs as fluorescent labels2. The effects of CdSe/ZnS quantum dots modified by streptavidin on PolymeraseChain ReactionPolymerase chain reaction (PCR) is an important nucleic amplification technology in modern biology. Since being invented by in1983, it has become apowerful tool in scientific research and promoted the development of life science. Withthe development of the technology, its improvement has never been stopped. Nowadays,nanomaterials were introduced into PCR to refine its performance.Quantum dots is a class of semiconductor material. It is concerned mainly due to itsexcellent optical property, while, its other properties such as surface group propert, areless concerned. Our study firstly introduces quantum dots modified by streptavidin toPCR system to study its effects on PCR performance.In this study we explore the effects of CdSe/ZnS modified by streptavidin on PCRperformance by conventional PCR technology and agarose gel electrophoresis. Firstly,We add different concentration of the quantum dots into PCR liquids to evaluate thespecificity of PCR. Secondly, the scope of the anneal temperature were evaluated byadding a certain concentration of the quantum dots into PCR liquids. Thirdly, thedifferent concentration of BSA was added into PCR liquis which has a certainconcentration of quantum dots. Finaly, the Taq polymerase from different companiescombined with different concentration of quantum dots into were added into PCRliquids to observe the effects.It is found that apropriate concentrationof CdSe/ZnS QDs modified by streptavidincan enhance the specificity, and widen the scope of the anneal temperature. BSA canreverse the effect of quantum dots on PCR. Excessive concentration of QDs can hinderthe performance of PCR. It is implied that the QDs modified by streptavidin can attactthe efficiency of PCR by interaction with Taq polymerase.