Novel multiplex and quantitative pathogen detection using stuffer-free MLPA-CE-SSCP
vi, 135 p.
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The rapid adaptation of microorganisms facilitated that emergence of new disease by expansion of international trade and environmental change. Therefore, it became important for new diagnostic tools to enable detection of broad range of infectious pathogens. In pathogen detection, multiplex, sensitive, rapid and quantitative detection are important criteria for early management of patients. However, the conventional culture-based methods take up to 7 days for results. On the other hands, PCR based detection methods are widely used in many applications because it can rapidly identify pathogenic species. However, PCR-based methods are suffered from limitations in multiplex and quantitative detection. To overcome the limitations, multiplex ligation-dependent probe amplification (MLPA) was introduced for simultaneously detect of several targets. Nevertheless, MLPA has limitations in specific probe designing that probe should include stuffer sequence to vary the length of target amplicons for length-dependent separation. In this thesis, a new method which stuffer-free MLPA based on a capillary electrophoresis–single strand conformation polymorphism (CE-SSCP) was developed to detect and discriminate various infectious pathogens. To improve stuffer-free MLPA-CE-SSCP method, we introduced various strategies to improve multiplexity, sensitivity, precision of analysis and rapid detection. First, to improve multiplexity the stuffer-free MLPA, strategies using efficient probe design method and separable probe design method were introduced. As a result, efficient probe design method was developed, and successfully detected and separated 10 pathogens in one reaction. Moreover, we solved design for separable probe sets using predictable free energyG value). Second, to improve the sensitivity of stuffer-free MLPA, whole genome amplification or target specific amplification was suggested and proven its effectiveness in a sensitive detection. By these approaches, the sensitivity was greatly improved, and at least 10 cells could be detected. Third, the quantification power was improved to be good enough for analysis of population of mixed pathogens. To accomplish this, optimization of reaction conditions was performed. Also, it was proven that the stuffer-free MLPA probes were very effective for a precise quantification because the short probe sets amplified equally. Lastly, to decrease total assay times, the reaction conditions were optimized and preconcentration method was introduced using antibody conjugated magnetic nanoparticle (Ab-MNP). The optimization of reaction condition effectively decreased total assay time which needs long reaction time, and the preconcentration reaction decreased enrichment time. By this approaches, the stuffer-free MLPA-CE-SSCP analysis time was decreased within 7 hours, and total assay time from samples could be decreased to 8 hours which is working hours in a day.