發(fā)布時間：2018-07-22 16:04

【摘要】：食源性疾病已成為全球性的公共衛(wèi)生問題,其中由食源性致病菌引起的食源性疾病發(fā)病率最高,對人類健康造成了巨大威脅。傳統(tǒng)的食源性致病菌檢測方法在檢測時間、靈敏度和特異性上存在一定的缺陷,不能滿足日益嚴格的檢測需要。因此,發(fā)展更為快捷,方便、靈敏度高和特異性好的檢測方法對保障食品安全具有重要意義。分子馬達是一種生物大分子,廣泛存在于細胞內,近年來得到國內外的普遍關注,其中旋轉分子馬達F_0F_1-ATPase作為生物傳感器被多次應用于食源性致病菌的檢測。該馬達具有靈敏度高、特異性強等特點。因此本研究以分子馬達F_0F_1-ATPase作為信號分子,以適配體作為識別分子,構建基于適配體識別的分子馬達生物傳感系統(tǒng),并應用于食源性致病菌的檢測,主要工作內容包括:首先,構建了一種基于適配體識別-F_0F_1-ATPase生物傳感檢測鼠傷寒沙門氏菌的方法。通過F_0F_1-ATPase表面ε亞基-ε亞基抗體-生物素-親和素-生物素化的鼠傷寒沙門氏菌適配體,將適配體固定于載色體表面;同時利用p H敏感型熒光染料F-DHPE監(jiān)測載色體膜外熒光強度的變化。當體系中存在沙門氏菌時,適配體特異性與沙門氏菌結合,使得F_0F_1-ATPase酶的負荷增加,引起旋轉速度下降,從而導致泵出載色體外的質子流速率下降,繼而p H值升高,最終導致熒光強度增強,通過監(jiān)測熒光強度的變化實現(xiàn)對沙門氏菌的檢測。結果顯示,沙門氏菌在10~1-10~4 cfu/m L范圍內與相對熒光強度具有良好的線性關系(y=1085.13x+363.98,R~2=0.9944),最低檢測限為10 cfu/m L。該方法已成功應用于牛奶實際樣品的檢測。其次,構建了以量子點為熒光指示物的F_0F_1-ATPase-適配體生物傳感檢測副溶血性弧菌的方法。通過F_0F_1-ATPase表面ε亞基-ε亞基抗體-生物素-親和素-生物素化的副溶血性弧菌適配體,將適配體固定于載色體表面;同時將p H敏感型量子點標記到載色體膜外,形成基于適配體識別的分子馬達生物傳感系統(tǒng)。當體系中存在副溶血性弧菌時,適配體特異性與副溶血性弧菌結合,使得F_0F_1-ATPase酶的負荷增加,引起旋轉速度下降,繼而引起H~+由載色體膜內泵出膜外的速率變慢,膜外的H~+濃度變小,p H值升高,量子點熒光強度增強,通過監(jiān)測熒光強度的變化實現(xiàn)對副溶血性弧菌的檢測。結果顯示,副溶血性弧菌在50-10~(6 )cfu/m L范圍內與體系相對熒光強度具有良好的線性關系(y=138.3x+13.05,R~2=0.9962),最低檢測限為5 cfu/m L。所建立方法已經(jīng)成功應用于市售蝦的檢測。
[Abstract]:Foodborne diseases have become a global public health problem, in which foodborne pathogens cause the highest incidence of foodborne diseases, which pose a great threat to human health. There are some defects in the detection time, sensitivity and specificity of the traditional methods for the detection of foodborne pathogenic bacteria, which can not meet the need of increasingly stringent detection. Therefore, it is important to develop a more rapid, convenient, sensitive and specific detection method to ensure food safety. Molecular motor is a kind of biological macromolecule, which widely exists in cells. In recent years, the molecular motor F0F1-ATPase has been widely used as a biosensor for the detection of foodborne pathogenic bacteria, among which the rotating molecular motor F0F1-ATPase has been widely concerned at home and abroad. The motor has the characteristics of high sensitivity and strong specificity. Therefore, in this study, the molecular motor FSP / F1-ATPase is used as signal molecule and the aptamer as recognition molecule to construct a molecular motor biosensor system based on aptamer recognition, and it is applied to the detection of foodborne pathogens. The main work includes: first of all, A biosensor method for detection of Salmonella typhimurium based on aptamer recognition-F0 F1-ATPase was developed. The aptamer of Salmonella typhimurium was immobilized on the surface of Color-carrying Salmonella typhimurium by using the antibody of 蔚 subunit 蔚 subunit on the surface of F0F1-ATPase-biotin-avidin-biotin-biotin aptamer of Salmonella typhimurium. At the same time, F-DHPE, a p-H sensitive fluorescent dye, was used to monitor the outside fluorescence intensity of the carrier. When salmonella was present in the system, the specific binding of aptamer to Salmonella caused the increase of the enzyme load of FSP _ 0F1-ATPase, which resulted in the decrease of rotation speed, which resulted in the decrease of proton velocity and the increase of pH value. Finally, the fluorescence intensity was enhanced, and the detection of Salmonella was realized by monitoring the change of fluorescence intensity. The results showed that there was a good linear relationship between the relative fluorescence intensity and salmonella in the range of 10 ~ 1010 cfu/m / L (yttrium 1085.13x 363.98 ~ 0.9944), and the detection limit was 10 cfu/m / L. The method has been successfully applied to the detection of milk samples. Secondly, a method for detecting Vibrio parahaemolyticus by using quantum dots as fluorescent indicator F0FSTs 1-ATPase- aptamer biosensor was constructed. The aptamer of Vibrio parahaemolyticus was immobilized on the surface of Vibrio parahaemolyticus by using the antibody of 蔚 subunit 蔚 subunit on the surface of F0F1-ATPase, and the pH-sensitive quantum dot was labeled outside the carrier membrane. A molecular motor biosensor system based on aptamer recognition is formed. When Vibrio parahaemolyticus exists in the system, the aptamer specificity binds to Vibrio parahaemolyticus, which increases the load of FSP _ 0F1-ATPase enzyme, decreases the speed of rotation, and then causes the rate of H ~ to pump out of the membrane of the chromogenic body to become slower. The fluorescence intensity of quantum dots increased with the increase of H concentration outside the film. The detection of Vibrio parahaemolyticus was realized by monitoring the change of fluorescence intensity. The results showed that the relative fluorescence intensity of Vibrio parahaemolyticus was linear in the range of 50-10 ~ (6) cfu/m L (yttrium 138.3x 13.05 cfu/m / L, 0.9962), and the lowest detection limit was 5 cfu/m / L. The established method has been successfully applied to the detection of commercial shrimp.
【學位授予單位】：江南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R155.3

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