本文選題：4懸浮芯片 + 食源性致病菌��； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2012年博士論文

【摘要】：食源性致病菌的污染不僅在世界范圍內(nèi)危害巨大，而且也嚴重影響我國進出口貿(mào)易，而致病菌的傳統(tǒng)檢測技術(shù)工作流程長、自動化水平低，難以適應(yīng)快速檢測的實際需求，其他常用的檢測方法又不能夠滿足多元檢測的要求。基于此，本研究旨在應(yīng)用高通量懸浮芯片技術(shù)，以六種常見的食源性致病菌包括大腸桿菌O157:H7、志賀氏菌、沙門氏菌、副溶血性弧菌、金黃色葡萄球菌和單核增生李斯特菌為檢測靶標物，建立一種新型快速、靈敏、高通量的檢測方法，以彌補現(xiàn)有方法不足，實現(xiàn)對食品中致病菌的快速、準確和高效多元檢測，進一步完善高通量懸浮芯片食品安全檢測技術(shù)平臺。 本研究以幾種常見的食源性致病菌為靶標物，基于核酸特異識別和抗體特異識別的原理，實現(xiàn)了對多種致病菌靶標物的同時多元檢測；通過對檢測過程的優(yōu)化和摸索實驗條件，進一步提高了懸浮芯片檢測的靈敏度、穩(wěn)定性；通過與PCR檢測方法和雙抗夾心ELISA法的比對實驗，確定了懸浮芯片法檢測的準確性，與常規(guī)檢測方法相比，懸浮芯片法檢測簡便、快捷，操作簡單，，結(jié)果重復(fù)性高，穩(wěn)定可靠。 本研究的研究內(nèi)容主要包括以下幾個方面： 1、基于致病菌16S rDNA的懸浮芯片多重檢測 以四種常見的食源性致病菌大腸桿菌、沙門氏菌、副溶血性弧菌和金黃色葡萄球菌為靶標物，以16SrRNA為靶基因，分別設(shè)計基于上述四種目標物的特異性引物及探針，將設(shè)計的探針與羧基化的聚苯乙烯微球偶聯(lián)，特異性地捕獲PCR擴增片段，從而達到檢測目標菌的目的。 對懸浮芯片檢測致病菌核酸的探針特異性及最佳雜交溫度進行了優(yōu)化，經(jīng)探針特異性驗證，所設(shè)計探針特異性較好，無明顯交叉反應(yīng)；對偶聯(lián)探針的微球與目標物雜交溫度條件進行了摸索，確定最佳雜交溫度為42℃。同時還對單通道和多通道檢測探針的靈敏度進行了驗證，最終單通道檢測探針靈敏度結(jié)果如下：大腸桿菌、沙門氏菌、副溶血性弧菌為5×10-6mmol/L，金黃色葡萄球菌為5×10-5mmol/L，多通道檢測探針的靈敏度結(jié)果如下：大腸桿菌、沙門氏菌、副溶血性弧菌和金黃色葡萄球菌均為1.25×10-4mmol/L。優(yōu)化了多重PCR檢測的樣品加入量，加入15μl多重擴增產(chǎn)物產(chǎn)生的熒光信號與單一PCR產(chǎn)物的熒光信號大致相當。將四種致病菌計數(shù)后分別添加到四種蔬菜中，利用懸浮芯片檢測，結(jié)果表明大腸桿菌在油菜中的檢測限和副溶血性弧菌在生菜中的檢出限均為103CFU/ml，沙門氏菌在黃瓜中和金黃色葡萄球菌在辣椒中的檢出限為100CFU/ml。 2、基于致病菌特有基因的懸浮芯片多重檢測 針對大腸桿菌O157:H7、志賀氏菌、沙門氏菌、副溶血性弧菌、金黃色葡萄球菌和單核增生李斯特菌的各自特有基因，設(shè)計引物，在引物擴增區(qū)內(nèi)設(shè)計能夠區(qū)別其他菌株的特異性探針，通過探針特異性驗證、探針靈敏度實驗、方法特異性實驗等證明該方法可以特異性的檢測六種致病菌，與常規(guī)PCR方法比較，檢測靈敏度明顯高于常規(guī)方法，多通道檢測探針靈敏度為1.6×10-6mmol/L,對單一PCR擴增產(chǎn)物的檢測最低檢出限為20-4×103CFU/ml，多重PCR擴增產(chǎn)物的檢測最低檢出限為1-10CFU/ml，不僅可以滿足日常食品安全檢測的要求，甚至可以滿足臨床樣品的檢測要求。 3、基于雙抗夾心法的致病菌免疫懸浮芯片檢測技術(shù)研究 分別制備了志賀氏菌、沙門氏菌和單核增生李斯特菌的多克隆抗體并生物素標記；建立了病原體蛋白懸浮芯片定量檢測方法；優(yōu)化了實驗的條件；討論了方法的特異性、靈敏度、檢測范圍、定量檢測能力，并與臨床常用的雙抗夾心ELISA方法進行了系統(tǒng)地對比。 本研究通過建立高通量致病菌檢測的懸浮芯片技術(shù)，通過對反應(yīng)條件的摸索，最終實現(xiàn)了可用于食源性致病菌的高通量檢測分析。檢測效果靈敏、快速、高效、穩(wěn)定，為食品中病原微生物快速檢測提供了新方法，具有廣闊的應(yīng)用和發(fā)展前景。
[Abstract]:The present study aims at the application of high - flux suspension chip technology in six common food - borne pathogenic bacteria including E . coli 157 : H7 , Shigella , Salmonella , Vibrio parahemolyticus , Staphylococcus aureus , and single - core hyperplasia Listerias to detect targets , and to establish a new rapid , sensitive and high - flux detection method to make up for the rapid , accurate and efficient multi - detection of pathogenic bacteria in food and further improve the technology platform for detecting food safety of high - flux suspension chips .

Based on the specific identification of nucleic acid and the principle of antibody - specific recognition , several common food - borne pathogenic bacteria were used to detect multiple pathogenic bacteria targets .
By optimizing and groping the test conditions , the sensitivity and stability of the suspension chip detection are further improved ;
Compared with the conventional detection method , the detection accuracy of the suspension chip method is determined by comparison with the PCR detection method and the double anti - sandwich ELISA method , and compared with the conventional detection method , the suspension chip method has the advantages of simple and convenient detection , rapid operation , simple operation , high repeatability and stable reliability .

The research contents of this study mainly include the following aspects :

1 . Multiple detection of suspension chip based on 16S rDNA of pathogenic bacteria

Four common food - borne pathogenic E . coli , Salmonella , Vibrio parahemolyticus and S . aureus were used as targets , and 16SrRNA was used as the target gene . Specific primers and probes based on the above four targets were designed respectively . The designed probes were coupled with carboxyl - functionalized polystyrene microspheres , and the PCR amplified fragments were specifically captured , so as to achieve the purpose of detecting the target bacteria .

The probe specificity and optimum hybridization temperature were optimized for the detection of the pathogenic bacteria nucleic acid in the suspension chip , and the specificity of the probe was verified by the specificity of the probe .
The results showed that the detection limit of E . coli , Salmonella , Vibrio parahemolyticus and Staphylococcus aureus were 1 . 25 脳 10 - 4 mmol / L .

2 . Multiple detection of suspension chip based on pathogenic bacteria specific gene

The detection limit of multiplex PCR products is 1 . 6 脳 10 - 6 mmol / L . The detection limit of multiplex PCR products is 1 . 6 脳 10 - 6 mmol / L . The detection limit of multiplex PCR products is 1 - 10CFU / ml .

Study on Detection Technique of Pathogenic Bacteria Immune Suspension Chip Based on Double Anti - Sandwich Method

Polyclonal antibodies and biotin - labeled antibodies were prepared for Shigella , Salmonella and Lister , respectively .
a quantitative detection method of the pathogen protein suspension chip is established ;
the conditions of the experiment are optimized ;
The specificity , sensitivity , detection range and quantitative detection ability of the method were discussed and compared with the commonly used double anti - sandwich ELISA method .

In this study , the high - throughput detection and analysis of food - borne pathogenic bacteria was achieved by establishing a suspension chip technology for high - flux pathogenic bacteria detection . The detection results were sensitive , rapid , efficient and stable , which provided a new method for rapid detection of pathogenic microorganisms in food , and had wide application and development prospects .
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R155.5

【參考文獻】

相關(guān)期刊論文 前10條

1 焦振泉,劉秀梅;16s rRNA序列同源性分析與細菌系統(tǒng)分類鑒定[J];國外醫(yī)學(xué)(衛(wèi)生學(xué)分冊);1998年01期

2 徐平,李同宇,秦莉;酶聯(lián)免疫吸附試驗在檢測志賀氏菌侵襲力中的應(yīng)用[J];臨沂醫(yī)專學(xué)報;2000年03期

3 黃寶華,陳慶森,龐廣昌;志賀氏菌研究及其快速檢測技術(shù)發(fā)展現(xiàn)狀[J];食品科學(xué);2004年11期

4 劉佩紅;屠益平;徐鋒;沈莉萍;;沙門氏菌檢測技術(shù)研究進展[J];上海畜牧獸醫(yī)通訊;2005年06期

5 許龍巖,李志勇,王志強,翁文川,謝鈞憲;PCR方法檢測志賀氏菌的研究[J];檢驗檢疫科學(xué);2003年05期

6 金周浩;宋達鋒;顧青;;副溶血弧菌毒力基因的檢測研究[J];中國食品學(xué)報;2008年03期

7 孫凱,汪謙;液相芯片技術(shù)研究應(yīng)用進展[J];中華實驗外科雜志;2005年05期

8 張曉峰,方維煥,江玲麗,杜華華;應(yīng)用聚合酶鏈反應(yīng)檢測食品中單核細胞增多性李斯特菌[J];中華預(yù)防醫(yī)學(xué)雜志;2003年03期

9 陳敏,周陶友,陳文昭,尹維佳,舒明蓉,呂曉菊;耐甲氧西林金黃色葡萄球菌感染的臨床和耐藥性[J];中華醫(yī)院感染學(xué)雜志;2004年02期

10 劉秀梅;食源性疾病監(jiān)控技術(shù)的研究[J];中國食品衛(wèi)生雜志;2004年01期

相關(guān)博士學(xué)位論文 前1條

1 蘇璞;牛奶中多種抗生素的懸浮芯片檢測技術(shù)研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2011年

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