茎环型引物—环介导等温扩增技术检测奶粉中单增李斯特菌的研究

发布时间：2018-07-14 11:09

【摘要】：单核细胞增生李斯特氏菌(Listeria monocytogenes,LM)简称单增李斯特菌,是一种能够引起人畜共患病的食源性致病菌,属李斯特菌属。广泛分布于自然界,食用了被单增李斯特污染的食物,可导致人和动物败血症、流产、脑膜炎等,病死率高达30%~70%,是人类最重要的食源性病原菌之一。单增李斯特菌的检测、鉴定以及防控技术已经受到极大的关注,其检测方法一直沿用过去传统的分离鉴定方法,己不能适应现代快速检测的要求。环介导等温扩增技术(Loop-mediated isothermal amplification, LAMP)是一种新颖的核酸体外扩增技术,具有简单、快速、敏感、特异性强的特点,已经应用于检测病原菌等诸多领域。该方法自身最大的局限性就是易出现假阳性。本研究利用LAMP技术检测单增李斯特菌,对LAMP引物进行设计、筛选及改进,选择带有一定茎环结构的引物,避免由于多条引物在同一条件下扩增,可能互补扩增而出现的假阳性,进一步提高LAMP检测的准确性,可称之为茎环型引物-LAMP(Stemloop-Primer Loop-mediated Isothermal Amplification,简称SLP-LAMP)。本试验以单增李斯特菌hlyA基因为靶基因,应用在线引物设计软件(https://primerexplorer.jp/lamp4.0.0/index.html)设计筛选出一套具有一定茎环结构的LAMP引物,建立了茎环型引物-环介导等温扩增技术检测单增李斯特菌的方法。对反应时间、反应温度、Mg2+浓度等扩增条件进行优化,确定25μL的SLP-LAMP反应体系为:内引物FIP和BIP各1.0μmol/L,外引物F3和B3各0.2μmol/L,dNTPs 0.4 mmol/L, MgCl2 4 mmol/L,10×Bst DNA聚合酶反应缓冲液2.5μL,8 U/μL的Bst DNA聚合酶0.6μL, DNA模板1.5μL,灭菌的去离子水补足体系。SLP-LAMP反应过程是首先在63℃水浴锅中反应50 min,然后将其放入80℃水浴锅中,水浴5 min终止反应,通过肉眼观察有无白色焦磷酸镁沉淀,即可判断是否发生了SLP-LAMP反应,亦可通过琼脂糖凝胶电泳证实反应是否发生。通过限制性内切酶ClaⅠ酶切扩增产物,测得酶切片段与理论预期值相符,验证了方法的正确性。本研究对3株单增李斯特菌、5株李斯特属的非单增李斯特菌株和14株其他食源性致病菌进行特异性试验。结果表明,3株单增李斯特菌结果为阳性,其他19株菌株为阴性。研究了6种从人工污染单增李斯特菌的奶粉中提取模板DNA的方法,结果表明SLP-LAMP检测对制备模板DNA方法的要求不高。本研究对SLP-LAMP法快速检测单增李斯特菌进行了研究,确定了检测纯培养物的灵敏度,人工污染检出限,并与普通PCR检测方法进行了对比。结果表明:SLP-LAMP检测单增李斯特菌的灵敏度为2.45×101 CFU/mL,其对人工污染奶粉样品的检出限为7.32×101 CFU/mL。采用试剂盒提取DNA,从样品处理到报告结果,耗时2 h。而对照,PCR检测单增李斯特菌纯培养物的灵敏度为2.45×10~3 CFU/mL,人工污染单增李斯特菌的检出限为7.32×10~3 CFU/mL。采用同样方法提取DNA,从样品处理到报告结果,耗时4 h。研究表明,本研究建立的SLP-LAMP法检测食品中单增李斯特菌的方法具有特异性强、灵敏度高,方便快捷、成本低等特点,并且通过大量的试验证明,筛选设计带有茎环型引物对避免反应过程中出现假阳性起到了一定的作用。为LAMP的检测提供了新的发展方向,该检测方法操作简单,不需要昂贵的仪器设备、繁琐的电泳过程,快速省时,更利于基层检验检疫机构的应用。
[Abstract]:Listeria monocytogenes (LM), called Listeria monocytogenes (LM), is a kind of food borne pathogenic bacteria that can cause zoonosis. It belongs to the genus Lester bacteria. It is widely distributed in nature. It has been eaten by single Lester contaminated food, which can cause septicemia, abortion, meningitis and so on. The mortality rate is up to 30%. ~70% is one of the most important foodborne pathogens of human being. The detection, identification and control technology of single increasing Lester bacteria have been paid great attention. The method of detection has always used the traditional methods of separation and identification, which can not meet the requirements of modern rapid detection.
Loop-mediated isothermal amplification (LAMP) is a novel amplification technique of nucleic acid in vitro. It has the characteristics of simple, rapid, sensitive and specific. It has been applied to the detection of pathogenic bacteria and many other fields. The maximum local limit of this method is the easy to appear false positive. This study uses LAMP technology to examine The LAMP primers were designed, selected and improved, and the primers with a certain stem ring structure were selected to avoid the false positive of multiple primers under the same condition and possible complementary amplification, which could further improve the accuracy of LAMP detection, which could be called the stem ring primer -LAMP (Stemloop-Primer Loop-mediated Isoth). Ermal Amplification, short for SLP-LAMP).
In this experiment, a set of LAMP primers with a certain stem ring structure were designed and screened using the hlyA gene of single increasing Lester bacteria as the target gene, and the method of detecting monocytogenes by the stem ring primer ring mediated isothermal amplification technique was established. The reaction time and reaction time were determined. The conditions of temperature, Mg2+ concentration and other amplification conditions were optimized, and the SLP-LAMP reaction system of 25 mu L was determined as: the primers FIP and BIP each 1 mol/L, the outer primers F3 and B3 0.2 micron each, dNTPs 0.4 mmol/L, MgCl2 4, 2.5 micron polymerase reaction buffer of 10, 1.5 micron, 1.5 micron of the template, and the sterilized deionized water. The system.SLP-LAMP reaction process is first to react 50 min in a 63 centigrade bath pot, then put it in a 80 C water bath, and the water bath 5 min terminates the reaction. By the naked eye, there is no white magnesium phosphate precipitation, can determine whether the SLP-LAMP reaction has occurred, and the reaction can be confirmed by agarose gel electrophoresis. The enzyme Cla I enzyme was amplified and the enzyme cut fragments were measured in accordance with the theoretical expectation, and the correctness of the method was verified. This study conducted specific tests on 3 strains of Lester bacteria, 5 non single Lester strains of Lester and 14 other foodborne pathogens. The results showed that 3 strains of Lester bacteria were positive, and the other 19 strains were positive. 6 methods of extracting template DNA from the milk powder of Lester bacteria by artificial contamination were studied. The results showed that the SLP-LAMP test was not very demanding for the preparation of the template DNA method.
In this study, the rapid detection of monocyculia monocytogenes by SLP-LAMP method was studied. The sensitivity of the pure culture, the detection limit of artificial contamination and the comparison with the common PCR detection method were determined. The results showed that the sensitivity of SLP-LAMP detected by SLP-LAMP was 2.45 x 101 CFU /mL, and the detection limit for the samples of artificial contaminated milk was 7.3. The 2 x 101 CFU/mL. used the kit to extract DNA, from the sample to the report result, the time consuming 2 h. and the control. The sensitivity of the pure culture of the single increasing Lester bacteria by PCR was 2.45 x 10~3 CFU/mL, the detection limit of the Artificially Polluted single Lester bacteria was 7.32 * 10~3 CFU/mL. using the same method to extract DNA, from the sample to the report result, and the time consuming 4 h..
The study shows that the method of SLP-LAMP method for detecting Lester bacteria in food has the characteristics of specificity, sensitivity, convenience, low cost and so on. And through a large number of experiments, it is proved that the selection of the primers with stem ring primers plays a certain role in avoiding the false positive in the reaction process. For the detection of LAMP For the new direction of development, the detection method is simple to operate, does not need expensive instruments and equipment, complicated electrophoresis process, fast and time-saving, and is more conducive to the application of basic inspection and quarantine organizations.
【学位授予单位】：河北农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：TS252.7;R155.5

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