食品中致病菌的检测方法研究进展

近年来，食品安全问题引起了世界各国的普遍重视。

据WHO初步统计，全世界每年的食源性疾病中，70%是由致病微生物引发的[1]。

食源性疾病和食品污染问题已成为世界范围内举足轻重的公共卫生问题。

在我国，每年上报的食物中毒人数逐年递增，且大部分是由食品中致病菌引起[2]。

十三五时期，史上最严的食品安全法提出了更高的要求和发展规划，食品安全已经成为国家安全的一部分。

其中，建立有效的食源性致病菌检测体系，快速准确完成食品中致病菌的检测，也成为保障食品安全和防止疾病传染的关键[3-5]。

下面主要对目前国内外食源性致病菌的检测方法进行归纳和概述。

1 免疫学检测免疫学检测依据免疫学理论和技术，基于抗原抗体反应，从而设计抗原、抗体、免疫细胞、细胞因子的检测分析方法。

微生物表面具有其特异的抗原，并能激发机体产生相应的特异性抗体；通过抗体结合表面抗原位点反应前后的信号变化，检测目标物；其中，借助标记物进行间接检测的研究，得到了快速的发展和检测效果的改善。

近年来，发展较快的免疫检测方法有如下四类。

1.1 酶联免疫吸附法酶联免疫吸附分析法的关键点是酶标记，利用标记酶对底物的高效催化性能，将信号进行放大，从而达到灵敏检测致病菌的目的。

在检测时，常借助抗原抗体间的特异性，通过三明治夹心结构将酶引入体系，此时载体上的酶量与待测菌的量呈现一定的比例。

加入酶的催化底物，酶与底物反应后所形成产物的量与待测菌的量呈现一定的关系，因此只要检测出产物的量就可以定性或定量检测致病菌。

由于酶有较高的催化效率，间接放大了免疫检测信号，该类检测方法具有较高的灵敏度。

1.2 荧光免疫检测法1941年，Coons等首次将荧光素用作标记构建检测体系。

经过发展，该类方法的标记物拓展到其它荧光活性分子，标记对象主要为抗原、抗体，在免疫反应结合后，通过荧光成像、荧光光度计等检测标记物的荧光信号，间接表示目标物含量。

该类方法在可视化检测领域具有很好的应用前景。

1.3 免疫胶体金标记分析法免疫胶体金标记技术主要包括胶体金光镜染色法、斑点金免疫渗滤法和胶体金免疫层析法。

它是一种将胶体金标记技术、层析分析技术以及免疫检测相结合的快速检测技术。

c# 图像识别其原理是依靠抗原与抗体的特异性结合，最终使胶体金变色，从而达到定性或定量检测目的。

在检测时，将抗原滴至加样孔膜上与金标抗体结合，在吸水材料的牵引下沿试条展开，到达检测线时由于特异性结合形成两个抗体结合一个抗原的复合物，由于金颗粒的沉淀，因此检测线呈红色。

未结合抗原的抗体行至对照线，与上边的抗体结合，使得对照的线呈现红色，出现两条红线。

若不含有目标致病菌则不发生结合，检测线不变红色，最终只出现一条红线。

1.4 免疫磁珠技术免疫磁珠分离技术将磁性分离与免疫反应相结合，从而构建免疫学检测技术；首先在磁珠上包被抗体，再与特定抗原发生特异性结合，将目标菌进行识别分离，最后磁力富集。

该类方法特异性强、灵敏度高、反应时间短。

如果将免疫磁珠分离技术与快速检测技术相结合，可以将致病菌检测时间由传统的几天缩短到几小时，因此，在食源性致病菌检测中，免疫磁珠技术具有广阔的发展空间。

2 分子生物学检测技术核酸生物分子作为生命体的基本单元，负责遗传信息的编码、传输及表达，作用非常重要。

它由核苷酸组成，根据化学组成不同分为核糖核酸（简称RNA）和脱氧核糖核酸（简称DNA）。

目前已有的核酸扩增技术分为两大类：靶核酸的直接扩增与信号放大扩增。

靶核酸直接扩增包括聚合酶链式反应、链替代扩增、连接酶链式反应和核酸依赖扩增、转录介导扩增和滚动环扩增；另一类是信号放大扩增：包括支链DNA、侵染探针和滚动环扩增等。

分子生物学方法在致病菌检测领域，得到了很好的应用[7]。

2.1 聚合酶链式反应（PCR）1985年，Mullis等人发明了PCR技术，相对而言，PCR技术具有特异性高、灵敏度好、操作简单、重复性好的优点。

PCR是一种DNA体外复制技术，通过复制放大，扩增目标互补DNA 片段，以微量的DNA为起点，获得大量的复制DNA。

PCR的基本原理和DNA复制过程相似，其特异性依赖于靶序列两端互补的寡核苷酸引物。

一般分为三个阶段：加热模板DNA发生变性；冷却使模板DNA与引物互补序列配对结合；在适度的温度下进行引物的延伸。

重复这三个过程能获得更多半保留复制链，从而能在短时间内将目的基因扩增放大几百万倍，PCR技术主要应用于单管PCR产物的实时荧光检测。

2.2 连接酶链式反应（LCR）1987年，Backman研究发明了LCR方法技术。

1991年，Backman和Barany分别用耐热DNA 连接酶延伸了LCR试验，为LCR的实用发展奠定了基础。

LCR技术依靠碱基的互补配对原理开展检测，在DNA连接酶的作用下，通过连接与模板DNA互补的两个相邻寡核苷酸链，进行DNA片段的快速扩增，从而复制出大量靶基因。

2.3 环介导等温扩增技术（LAMP）2000年，Notomi等首次提出LAMP技术，LAMP技术依靠能对靶序列上的6个独立区域特异性识别的4种引物，及具有链置换活性的DNA聚合酶，通过循环链置换反应，达到靶序列的扩增。

反应包括两个阶段，循环扩增始发物的形成与循环延伸。

首先，由外部引物扩增出内部引物所需的模板，接着内部引物对靶基因片段进行引导合成。

如此循环反应最终形成带有类似花椰菜的茎-环结构，该法可在短时间内实现109—1010倍的扩增。

Malcolm等首次结合多重PCR技术和LAMP方法，构建了副溶血性弧菌的定量检测体系[9]。

Lee结合LAMP 和免疫层析技术，以未前处理的全血和牛奶为检测对象，实现了金黄色葡萄球菌和大肠杆菌O157：H7的检测[10]。

LAMP方法是一种快速简便、省时省力、特异性好、灵敏的致病菌检测技术。

2.4 滚动环式扩增（RCA）RCA是一种较新的恒温核酸扩增技术，原理类似于噬菌体复制过程，它以环形DNA为模板，通过一个与部分模板DNA互补的较短DNA引物，在酶的催化下将dNTPs转变成包含无数个能与模板互补的单链DNA片段，因此，它既可以进行靶核酸基因的扩增，又可以进行型号的放大扩增。

滚动环式扩增分为两种形式：线性扩增和指数扩增。

线性扩增是指引物结合到模板DNA后，在酶的作用下，产物具有无数个能与模板互补的线状单链DNA片段，线性RCA 仅仅适用于一些具有环状核酸的病毒、染色体的扩增。

RCA可以使产物呈线性增加，因此可以用于靶核酸的定量检测。

指数扩增的原理与线性扩增基本相同，只是采用与模板DNA序列完全相同的引物，在酶作用下延伸，其产物作为探针模板；因此，在很短的时间里可以使产物呈现指数递增，指数扩增常用于非环状DNA的扩增。

2.5 基因芯片技术DNA微阵列又称DNA阵列、DNA芯片、基因芯片，最早出现在80年代中期。

基因芯片的基本原理是杂交测序法，一般来说基因芯片是指带有适配体涂层的特殊物体；具体地说，基因芯片能够识别特定基因表达、基因序列，最常见的基因芯片是指将单链DNA探针固定在其上的特殊玻璃片。

近年来，有越来越多的研究团队运用基因技术检测食源性致病菌，Seung Jun Oh等设计制备了圆形微流控芯片，该芯片借助LAMP和铬黑T光学手段实现了多种致病菌的甄别和检测[11]。

YongTae Kim等结合固相萃取、聚合酶链式反应和免疫层析技术，构建了多种致病菌的光学检测体系，检测过程可在55分钟内完成，检测限达5 cell[12]。

基因芯片技术能够获得目标物详细的基因信息，包括相关识别因子、类型、致病因子和抗生素抗药性等。

但它存在成本高、检测能力有限、重现性低等缺点，因此，代写毕业论文基因芯片技术在致病菌检测中的运用还需要进一步研究[13]。

In recent years, the problem of food safety has attracted the attention of all countries in the world. According to WHO preliminary statistics, the world's annual food borne disease, 70% is caused by pathogenic microorganisms [1]. Food borne diseases and food pollution has become a worldwide public health problem. In our country, the annual report of the number of food poisoning is increasing year by year, and most of them are caused by the bacteria in food [2]. During the period of 45, the history of the most stringent food safety law proposed higher requirements and development planning, food safety has become a part of the national security. Among them, the establishment of effective food borne pathogens detection system, rapid and accurate detection of pathogenic bacteria in food, but also become the key to ensuring food safety and prevent disease transmission [3-5]. Here are the main methods for the detection of foodborne pathogens at home and abroad are summarized and summarized.1 immunological testBased on the theory and technology of immunology, antigen antibody reaction was designed, which was designed to detect antigen, antibody, immune cell and cytokine. Microbial surface with its specific antigen, and can stimulate the body to generate the corresponding specificity antibody; by antibody binding surface antigen reaction sites before and after the signal changes, to detect the target; which, with markers for indirect detection research, has been the rapid development and the detection effect improved. In recent years, the rapid development of the immune detection methods are as follows: four.1.1 enzyme linked immunosorbent assayEnzyme linked immunosorbent assay is the key point of the enzyme marker, the use of marker enzyme on the substrate of high efficiency catalytic performance, the signal amplification, so as to achieve the purpose of sensitive detection of pathogenic bacteria. When detecting, the enzyme is introduced into the system through the sandwich structure by the specificity of the antigen antibody, and the amount of the enzyme and the amount of the bacteria to be measured is proportional to the amount of the enzyme. The amount of the product formed by the reaction between enzyme and substrate is related to the amount of the substrate to be tested, so as long as the amount of the product can be detected qualitatively or quantitatively. Because the enzyme has high catalytic efficiency, the immune detection signal is amplified, and the detection method has high sensitivity.1.2 fluorescence immunoassayIn 1941, Coons, for the first time, was used as a marker to construct the detection system. After development, markers of this method extended to other fluorescent molecules, object is marked mainly antigen, antibody, combined in the immune response, by fluorescence imaging, fluorescence photometry meter etc. marker detection of fluorescence signals, indirectly indicating a target content. This kind of method has a very good application prospect in the field of visual inspection.1.3 immuno colloidal gold labeling analysisImmune colloidal gold labeling technique including light microscopy colloidal gold staining method, dot immunogold filtration assay and colloidal gold immune chromatography. It is a kind of rapid detection technology, which combines colloidal gold labeling technology, chromatography analysis technology and immunoassay. C# image recognition of the principle is to rely on the specificity of antigen and antibody binding, and ultimately make colloidal gold color change, so as to achieve the purpose of qualitative or quantitative detection. In the detection, antigen drops to kind of film hole with gold labeled antibody binding and absorbent material traction along the test strip expansion, the detection line arrived due to specific binding to form two antibody binding to an antigen complexes, due to the precipitation of gold particles. Therefore, the detection of line is red. Unbound antigen for antibody to control line, from the top of the antibody binding, the control line appear red, two red lines. If the target does not contain pathogenic bacteria are not combined, the detection line is not the same red, and ultimately only a red line.1.4 immunomagnetic beadsImmunomagnetic separation technology combining with the immune response to magnetic separation, so as to construct the immunological detection technology; first the beads package by antibodies, and specific antigen occurred specific binding, target bacteria identification and separation, the magnetic enrichment. This kind of method has the advantages of high specificity, high sensitivity and short reaction time. If combining immunomagnetic separation technology and rapid detection technology, will cause the pathogen detection time by traditional days is reduced to a few hours. Therefore, in foodborne pathogenic bacteria detection, immune magnetic bead technique has broad space for development.2 molecular biology detection technologyNucleic acid biological molecules as the basic unit of life, is responsible for the coding, transmission and expression of genetic information, the role is very important. It consists ofnucleotides, according to the chemical composition of the different divided into RNA (referred to as RNA) and DNA (referred to as DNA). At present, there are two kinds of nucleic acid amplification techniques: direct amplification of target nucleic acids and amplification of signal amplification. Target direct nucleic acid amplification polymerase chain reaction chain, alternative amplification and ligase chain reaction and nucleic acid dependent amplification, transcription mediated by amplification and rolling circle amplification; another kind is signal amplification: including branched DNA, infection probe and rolling circle amplification and. The molecular biology method has been applied to the detection of pathogenic bacteria, and it has been well applied in the field of [7].2.1 polymerase chain reaction (PCR)In 1985, Mullis, who invented the PCR technology, relatively speaking, PCR technology with high specificity, good sensitivity, simple operation, good repeatability. PCR is a DNA in vitro replication technology, through the replication amplification, amplification target complementary DNA fragments, to trace the DNA as a starting point, to obtain a large number of replication DNA. The basic principle of PCR is similar to that of DNA replication process, and its specificity is dependent on the complementary oligonucleotide primers. Generally divided into three stages: the heating template DNA denaturation; cooling to make the template DNA.。