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|標題:||Flowomics- Technology Development of High Throughput Microbial Identificaiton and Quantifiation
To date, rapid and accurate characterization of a microbial ecosystem is still an unreachable dream. There is no technology available to acquire the following three types of information at the same time: (1) “who are there?” (2) “how many are there of each species?”, and (3) “what is each cell doing?”. Making available this information will not only benefit fundamental scientific research in microbial ecology but also improve the optimization of biological processes in all microbiologically related areas, such as biological processes in food industry, drug biosynthesis, diagnosis of virulent strains of specific viruses or bacteria, biodegradation of contaminants in soils and groundwater, and biological treatment of wastewater, etc.For microbial identification and quantification, culture-based methods are too time-consuming and pose selection pressure (bias) on microorganisms. PCR-type diagnosis is used to amplify biological biomarkers, such as 16S rDNA, intergenic spacer genes or other specific genetic makers, and then to identify and quantify the amplified markers. However, due to possible bias caused by primers and chimeric sequence formation, PCR may result in unexpected results. Whole cell type analyses, such as FISH, are still time-consuming and target-limiting. Recently, FISH-MAR has been shown to be promising in linking microbial community structure with functional information but it can only offer observation no more than seven bacterial populations due to the availability of fluorophores. The so-called isotope array can potentially detect thousands of species at a time but still cannot provide fully quantitative results and the result was impaired by more than 200 false-positives in a single test. Due to the lack of an effective measure to identifying all bacterial species in a sample, other methods were developed to observe the abundance and structure of a microbial ecosystem. However, they still cannot provide the aforementioned three types of information at the same time.Here, we propose a novel approach to rapidly and accurately identify and enumerate bacteria by integrating fluorescent molecular beacon with microfluidic technology, bacterial bioinformatics, single molecule detection in microdroplets, and statistical matching. It is estimated that this proposed prototype, one can easily identify 9 different bacterial species by using two fluorescent wavelengths (or 256 bacteria with 4 wavelengths). With single molecule detection, this approach will guarantee full quantitative results with much shorter turn-around time. This critical study will provide the base for a full spectrum platform that can identify, quantify, and define functional roles of thousands of bacterial cells instantaneously.
環境微生物與微生物生態學 (Microbial Ecology) 領域中始終存在一個無法解答的問題，即在任何環境樣品中「有哪些微生物存在？」、「各種各有多少？」及「每一個細胞正在做什麼？」。此資訊不僅有助於解決環境微生物學與微生物生態學之基礎問題，更可應用於其他各種與微生物相關之領域，如食品加工、製藥之生物合成、致病性菌株鑑定、廢水之生物處理與土壤及地下水污染生物整治等。在微生物鑑定上，培養基鑑定法既費時且靈敏度有限。近來，因分子生物技術進步，可以聚合酶連鎖反應將特定之生物標記放大並以分子探針及螢光定量，如16S rDNA、intergenic spacer genes 或是其他特定之基因標記，但仍費時並屬間接定量，且引子之使用可能導致結果偏歧。全細胞式定量方法，雖可直接定量並觀察若干功能性基因，但仍費時且靈敏度偏低。雖然還有其他針對生態系龐雜度(Diversity)或豐盛度(Abundance)之定量方法.仍無法快速鑑定定量各種微生物及定義其所處生態系中之功能角色。微流體生物晶片具有高分析靈敏度、分析速度快、樣品及試劑少、高度可攜性、減少人為誤差等優點。此外，可將分析化學實驗室中之儀器設備微小化並製作在平面化的晶片上，具有樣本進樣（Sampling）、混合（Mixing）、傳輸（Sample Transport）、反應（Reaction）、分離（Separation）以及檢測(Detection)等多項功能；本研究藉由微機電工程中之微影製程及軟微影製程進行微流體生物晶片之光罩設計、母模製作、與實驗晶片開發，製作本研究專用之微流體晶片。為解決上述環境微生物與微生物生態學之難題，茲提出以基因體中去氧核糖核酸中之重複片段(genomic DNA repeat sequences)為鑑定標的，並結合微流體生物晶片、微水珠形成、單一細胞封裝、細胞快速溶解、特殊分子探針設計、微水珠中快速雜合、單分子偵測、光學信號快速擷取及電腦統計方法進行超快速高通量鑑定與定量。
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