流式質體儀---超高速之微生物鑑定定量研究

Abstract

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 will also improve the optimization of biological processes in all microbially related areas, e.g., biological processes in food industry, drug biosynthesis, early diagnosis of virulent strains of specific viruses or bacteria, biodegradation of contaminants in soils and groundwater, and biological treatment of wastewater.For microbial identification and quantification, culture-based methods are too time-consuming and pose selection pressure (bias) on microorganisms1. 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 markers2-5. However, due to possible bias caused by primers and chimeric sequence formation, PCR may result in unexpected results6-8. 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 fluorophores9. 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 test9, 10. 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 time11-15.Here, we propose a novel approach to rapidly and accurately identify and enumerate bacteria by integrating bacterial genomic repeats information with microfluidic device,16-18 and single molecule detection in droplets19-21. It is estimated that by using this proposed prototype, one can easily identify 9 different bacterial species by using two fluorescence detectors. With single molecule detection, this approach will guarantee full quantitative results with much shorter turn-around time. This critical result 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未能有效解答的問題：即在一環境樣品中「有何種微生物存在？」、「各種各有多少？」及「各個細胞各在做什麼？」。此方面之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，如食物加工、製藥及生物合成、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功能角色。為解決此一難題，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、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。