研發檢測食源性致病菌及毒素之分析方法-總計畫及子計畫二：開發適合分析食源性病菌及毒素之親和分析方法( I )

Abstract

食品可能在加工製造或儲藏過程中遭受到細菌和毒素污染，因此如何做好食品品質 控管以確保食品之品質是相當重要的一環。發展快速分析方法可用以監測、篩選、及確 認食品中的污染物有實際之必要性，而且一旦爆發任何食品污染事件，需要有適合分析 方法用於快速確認食品污染物以防止進一步之不良影響情事發生，如最近在海地爆發的 霍亂感染，短短幾個月間已奪走數千條人命。但是食品通常成份相當複雜且含複雜基質， 一般如不事先進行任何前處理步驟，通常無法獲得有用的污染物的分析資訊，本計劃的 目標是發展以親和為概念的靈敏檢測方法，可用於篩選及確認食品中細菌及毒素。我們 計畫使用的策略是以碳水化合物分子為探針，用以選擇濃縮食品中可以與特定醣分子有 親和作用力的目標致病菌和毒素，計劃中將首先使用凝集素-即刀豆球蛋白A 為測試樣 品，再進一步應用於食源性凝集素蛋白類型的毒素如蓖麻毒蛋白及細菌表面具凝集素類 分子的致病菌如綠膿桿菌/豬鏈球菌為樣品，而以螢光光譜法和質譜法等可以提供定量 和定性分析的資料的分析技術為偵測方法。 此一跨領域研究計畫將由兩個互有相關的子計畫組成：第一個子計畫為”合成對食 源性致病菌和毒素有親和作用力的功能化碳水化合物分子”，此子計畫一將由交通大學 應用化學系蒙國光博士領導下的研究團隊負責執行，此子計畫執行重點為合成對目標致 病菌和毒素具辨識能力之探針分子，他們將合成包括多價醣樹枝狀分子及共軛醣衍生物 等之碳水化合物分子用以辨識食源性致病菌和毒素;第二個子計畫為”開發適合分析食 源性病菌及毒素之親和分析方法”，此子計畫二將由交通大學應用化學系陳月枝博士領 導下的研究團隊負責執行，執行重點為使用功能化碳水化合物分子為探針分子發展可快 速篩選及確認食源性致病菌及毒素的分析方法。功能化碳探針分子則將以醣樹枝狀分子 及修飾有醣化合物的奈米粒子為探針基材，並以發展可簡易利用肉眼快速辨識污染物是 否存在，而以螢光光譜法和質譜法為定量及定性的偵測方法。除此之外，專長研究領域 在細菌致病機轉的彭慧玲教授(交通大學生物科技系)將為本計劃之共同主持人，協助我 們在細菌偵測的研究。 我們期望藉此跨領域計畫執行可發展適用於快速檢測食源性致病菌和毒素的方法， 因而有利於快速篩選這些食品中的污染物。

Food products are easily contaminated by microorganisms and toxins when they are processed improperly. Quality assurance and control can reduce the chances of contracting harmful contaminants in food products. Thus, developing rapid detection methods for screening of target contaminants is relevant and urgently required. Beyond the practical reasons, it is also significant to confirm the identity of contaminates in a short period of time once any foodborne illness outbreak takes place owing to the presence of pathogens and toxins in food products. The consequence of food contamination would be devastating such as the recent cholera outbreak in Haiti took the lives of thousands in few months. However, food samples or products are complex mixtures. Thus, without any pretreatment processes, it is almost impossible to obtain useful analytical information of contaminants by directly analyzing samples. Therefore, the aim of this proposal is to develop fast, reliable, and sensitive methods for detection of foodborne contaminants including pathogens and toxins. The main strategies we plan to use herein are based on the specific binding of carbohydrate ligands to target pathogens and toxins capable of interacting with specific carbohydrate molecules from complex samples. As a model study, detection of lectin proteins-concanavalin A will be firstly examined by using this method. Subsequent studies would examine the potentially lethal lectin type toxins such as ricin and pathogens such as Pseudomonas aeruginosa and Streptococcus suis, whose outer membranes contain galactophilic lectins capable of interacting with specific glycans. Fluorescence spectroscopy and mass spectrometry for quantitative and qualitative analysis will be utilized as the detection tools. Naked eye detection method for rapid screening of the target contaminants is also going to be established. This interdisciplinary research project is composed of two mutually related sub-projects: (1) Synthesis of structurally defined functional carbohydrates for probing foodborne pathogens and toxins, led by Dr. K. K. T. Mong from the Department of Applied Chemistry at the National Chiao Tung University. His group will focus on syntheses of functionalized carbohydrate ligands including glycan-derivatives and glycodendrimers as the probe molecules for target species. (2) Exploration of affinity-based assays for foodborne pathogens and toxins, led by Dr. Yu-Chie Chen from the Department of Applied Chemistry at National Chiao Tung University, puts efforts on the exploration of suitable assays for foodborne contaminants. Functional molecule/nanomaterial-based affinity approaches will be developed for the detection of target species. Prof. Hwei-Ling Peng, whose research expertise is in bacterial pathogenesis, from the Department of Biological Science and Technology at the National Chiao Tung University will be the co-PI of this proposal. With her joining, we will be benefit quite a bit in the topic of bacterial detection mainly conducted in this proposal. We expect that new types of carbohydrate-based affinity approaches for rapid detection of target bacteria and toxins will be developed in this interdisciplinary project. The developed analytical methods will be beneficial for rapid on-site screening and characterization of carbohydrate-recognized foodborne pathogens and toxins.