食源性致病菌高通量悬浮芯片检测技术研究

发布时间：2018-06-13 06:48

本文选题：4悬浮芯片 + 食源性致病菌　；参考：《中国人民解放军军事医学科学院》2012年博士论文

【摘要】：食源性致病菌的污染不仅在世界范围内危害巨大，而且也严重影响我国进出口贸易，而致病菌的传统检测技术工作流程长、自动化水平低，难以适应快速检测的实际需求，其他常用的检测方法又不能够满足多元检测的要求。基于此，本研究旨在应用高通量悬浮芯片技术，以六种常见的食源性致病菌包括大肠杆菌O157:H7、志贺氏菌、沙门氏菌、副溶血性弧菌、金黄色葡萄球菌和单核增生李斯特菌为检测靶标物，建立一种新型快速、灵敏、高通量的检测方法，以弥补现有方法不足，实现对食品中致病菌的快速、准确和高效多元检测，进一步完善高通量悬浮芯片食品安全检测技术平台。本研究以几种常见的食源性致病菌为靶标物，基于核酸特异识别和抗体特异识别的原理，实现了对多种致病菌靶标物的同时多元检测；通过对检测过程的优化和摸索实验条件，进一步提高了悬浮芯片检测的灵敏度、稳定性；通过与PCR检测方法和双抗夹心ELISA法的比对实验，确定了悬浮芯片法检测的准确性，与常规检测方法相比，悬浮芯片法检测简便、快捷，操作简单，，结果重复性高，稳定可靠。本研究的研究内容主要包括以下几个方面： 1、基于致病菌16S rDNA的悬浮芯片多重检测以四种常见的食源性致病菌大肠杆菌、沙门氏菌、副溶血性弧菌和金黄色葡萄球菌为靶标物，以16SrRNA为靶基因，分别设计基于上述四种目标物的特异性引物及探针，将设计的探针与羧基化的聚苯乙烯微球偶联，特异性地捕获PCR扩增片段，从而达到检测目标菌的目的。对悬浮芯片检测致病菌核酸的探针特异性及最佳杂交温度进行了优化，经探针特异性验证，所设计探针特异性较好，无明显交叉反应；对偶联探针的微球与目标物杂交温度条件进行了摸索，确定最佳杂交温度为42℃。同时还对单通道和多通道检测探针的灵敏度进行了验证，最终单通道检测探针灵敏度结果如下：大肠杆菌、沙门氏菌、副溶血性弧菌为5×10-6mmol/L，金黄色葡萄球菌为5×10-5mmol/L，多通道检测探针的灵敏度结果如下：大肠杆菌、沙门氏菌、副溶血性弧菌和金黄色葡萄球菌均为1.25×10-4mmol/L。优化了多重PCR检测的样品加入量，加入15μl多重扩增产物产生的荧光信号与单一PCR产物的荧光信号大致相当。将四种致病菌计数后分别添加到四种蔬菜中，利用悬浮芯片检测，结果表明大肠杆菌在油菜中的检测限和副溶血性弧菌在生菜中的检出限均为103CFU/ml，沙门氏菌在黄瓜中和金黄色葡萄球菌在辣椒中的检出限为100CFU/ml。 2、基于致病菌特有基因的悬浮芯片多重检测针对大肠杆菌O157:H7、志贺氏菌、沙门氏菌、副溶血性弧菌、金黄色葡萄球菌和单核增生李斯特菌的各自特有基因，设计引物，在引物扩增区内设计能够区别其他菌株的特异性探针，通过探针特异性验证、探针灵敏度实验、方法特异性实验等证明该方法可以特异性的检测六种致病菌，与常规PCR方法比较，检测灵敏度明显高于常规方法，多通道检测探针灵敏度为1.6×10-6mmol/L,对单一PCR扩增产物的检测最低检出限为20-4×103CFU/ml，多重PCR扩增产物的检测最低检出限为1-10CFU/ml，不仅可以满足日常食品安全检测的要求，甚至可以满足临床样品的检测要求。 3、基于双抗夹心法的致病菌免疫悬浮芯片检测技术研究分别制备了志贺氏菌、沙门氏菌和单核增生李斯特菌的多克隆抗体并生物素标记；建立了病原体蛋白悬浮芯片定量检测方法；优化了实验的条件；讨论了方法的特异性、灵敏度、检测范围、定量检测能力，并与临床常用的双抗夹心ELISA方法进行了系统地对比。本研究通过建立高通量致病菌检测的悬浮芯片技术，通过对反应条件的摸索，最终实现了可用于食源性致病菌的高通量检测分析。检测效果灵敏、快速、高效、稳定，为食品中病原微生物快速检测提供了新方法，具有广阔的应用和发展前景。
[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 .
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R155.5

【参考文献】