【作者】 王鹏；

【导师】 张伟；

【作者基本信息】 河北农业大学 ， 农产品加工及贮藏工程， 2011， 硕士

【摘要】 病原微生物与食品污染构成了一个巨大并不断扩大的世界性公共卫生问题。我国食源性疾病监测网的统计数字显示:近十年来,由病原微生物引起的食源性疾病事件和涉及的人数最多占46.4%,其中变形杆菌属占11.3%,普通变形杆菌和奇异变形杆菌是引起食物中毒的主要变形杆菌。因此,普通变形杆菌和奇异变形杆菌的检测、鉴定以及防控技术已经受到极大的关注。变形杆菌属的检测方法一直沿用过去传统培养法,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,简称LAMP）是日本学者Notomi等于2000年发明的一种全新的核酸扩增方法。该技术利用能识别靶序列上6个位点的4个特殊的引物和一种具有链置换活性的DNA聚合酶,在恒温条件下,特异、高效、快速地扩增核酸。在1 h内扩增效率可达到10⁹-10¹0个数量级,扩增产物是一系列反向重复的靶序列构成的茎环结构和多环花椰菜样结构的DNA片段混合物,电泳后在凝胶上显现出由不同大小的区带组成的阶梯式图谱。另外,在反应中添加特异性环引物（loop primer）能够把扩增时间缩短到原来的一半,使反应速率极大地得到提高。近年来LAMP技术以其特异性强、等温、灵敏、操作简单、产物易检测等优点已经应用于食品安全检测领域的多个方面。本研究针对变形杆菌属atpD基因序列（AX10⁹601）进行分析,设计6条引物（2条内引物、2条外引物、2条环引物）,对普通变形杆菌和奇异变形杆菌进行检测,优化反应条件,验证检测的特异性和灵敏度,应用于食品样品的直接检测,并用限制性内切酶Psp1406Ⅰ酶切扩增产物,观察酶切片段大小,验证方法的正确性,建立了变形杆菌属的LAMP检测方法。为了确立最佳反应条件我们对LAMP反应体系进行了优化。实验表明温度是LAMP的最大影响因素,而镁离子和引物浓度比对反应影响不大。从而获知外环内引物浓度比为1:2:6、Mg²⁺添加浓度为2.0 mmol/L、反应温度为61℃、反应时间为50 min时检测最灵敏。为评价该技术的特异性,我们同时对1株普通变形杆菌、1株奇异变形杆菌和13株其它肠杆菌致病菌进行特异性试验。结果表明仅变形杆菌属DNA扩增产物在电泳后出现特异的梯状条带,进一步对该扩增产物进行酶切,结果也与预期相符。这表明LAMP技术对变形杆菌属DNA的扩增是特异的。我们对原始模板进行梯度稀释来测试其敏感性,实验结果表明LAMP技术可以稳定检出变形杆菌DNA的下限为5.4 CFU/mL。应用LAMP方法对猪肉样品进行变形杆菌DNA检测,并将结果与普通PCR法相比较。结果显示,LAMP检测猪肉中变形杆菌的检出限为15 CFU/mL,对照PCR检测猪肉中变形杆菌的检出限为150 CFU/mL。LAMP方法比PCR方法检出限高了10倍,时间也缩短了一半。因此,我们的实验表明LAMP技术用于食品中变形杆菌属DNA检测具有很高的特异性和敏感性。并且LAMP所具有操作简单快速,结果易判断和不需要复杂技术及设备的低成本优势也是PCR所不能及的。如果进一步优化、完善和推广,我们相信该技术将会在食品感染变形杆菌的预防上发挥重要作用。更多还原

【Abstract】 Pathogenic microorganisms and food contamination constitute a large and growing public health problem worldwide. Monitoring network of food-borne disease statistics: over the past decade, caused by the pathogenic microorganisms and foodborne disease events involving the largest number of 46.4%, which accounted for 11.3% of Proteus, Proteus vulgaris and Proteus mirabilis Is the major cause of food poisoning Proteus. Therefore, Proteus vulgaris and Proteus mirabilis in the detection, identification, and prevention and control technology has been of great concern. Proteus detection method has been used in traditional culture, but have been unable to meet the modern requirements of rapid detection.Loop-mediated isothermal amplification （Loop-mediated isothermal amplification, referred to as LAMP） is equal to 2000, the Japanese scholar Notomi invented a new method of nucleic acid amplification. The technology used to identify the target sequence sites on the 6 4 specially designed primers and a DNA with strand displacement activity of polymerase, at a constant temperature, specific and efficient, rapid amplification of nucleic acids. In amplification efficiency can be achieved within 1h 10⁹-10¹0 orders of magnitude, is a series of inverted repeat amplification target sequence stem-loop structure and composition of multi-ring structure of cauliflower-like mixture of DNA fragments, after gel electrophoresis showing zones of different sizes consisting of stepped patterns. In addition, to add specific ring in the reaction primers （loop primer） able to amplify shorter by half, so that the reaction rate greatly improved. LAMP technology in recent years for its specificity, so temperature sensitive, simple, easy to test the advantages of the product has been used in the field of food safety in many areas.In this study, Proteus atpD gene sequence （AX10⁹601） for analysis, design, six primers （two inner primers, two outer primers, two loop primers）, and Proteus vulgaris and Proteus mirabilis were detected, optimal reaction conditions to verify the specificity and sensitivity of detection, applied to the direct detection of food samples, and using restriction enzyme digested PCR products Psp1406Ⅰobserved fragment size, the correctness of authentication methods, the establishment of the LAMP detection of Proteus methods. To establish the optimal reaction conditions we LAMP reaction were optimized. Experiments show that the maximum temperature is the LAMP factors, while the concentration of magnesium ions and the ratio of primer have little effect on the reaction. Informed of the outer ring to inner primer concentration ratio of 1:3:6, Mg²⁺ concentration was 4 mmol/L, reaction temperature is 61℃, reaction time was 50 min when the most sensitive test. To evaluate the specificity of the technology, we also on a Proteus vulgaris, a Proteus mirabilis and 13 strains of other pathogenic E.coli-specific test. The results showed that Proteus only in the DNA amplification products after electrophoresis specific laddering, the PCR products were digested further, the results are in line with expectations.This shows that the LAMP technology Proteus is a specific DNA amplification. Our serial dilution of the original template to test the sensitivity and experimental results show that the detection of LAMP technology can stabilize the lower limit of Proteus DNA 5.4 CFU/mL. Application of LAMP method Proteus pork DNA samples were detected, and the results compared with the normal PCR wears. The results showed that, LAMP detected Proteus pork detection limit of 15 CFU/mL, control PCR detection of pork in the detection limit of Proteus 150 CFU/mL. LAMP method than the PCR detection limit 10 times higher, and time is reduced by half.Therefore, our experiments show that the LAMP technology for the food Proteus DNA detection with high specificity and sensitivity. The LAMP is simple and rapid, easy to judge and does not require sophisticated technology and equipment is low-cost advantages of PCR can match. If further optimization, improvement and promotion, we believe that the technology will be infected with Proteus in food play an important role in prevention.更多还原