【作者】 钱翔宇；

【导师】 张伟；

【作者基本信息】 河北农业大学 ， 微生物学， 2009， 硕士

【摘要】 普通变形杆菌（Proteus.vulgaris）是一种食源性致病菌,广泛存在于自然界,存在于污水,土壤和垃圾中,也存在于人和动物的肠道中,是一种条件致病菌,容易污染肉类和蛋类等动物性食品,可导致胃肠炎,脑膜炎,败血症等疾病。传统检测变形杆菌属的方法操作繁琐,检测时间长,至少需要48-72h。不能满足食品中致病菌快速、灵敏的检测需要。因此需要研究变形杆菌的检测方法,为日常食品卫生检测提供快速检验的方法。环介导等温扩增（loop-mediated isothermal amplification,简称LAMP）是由日本学者Notomi等人于2000发明的一种全新的核酸扩增方法。它采用能特异识别靶序列上6个位点的4条引物及一种具有链置换活性的DNA聚合酶（BstDNA polymerase）,在恒温条件（60～65℃）下,特异、高效、快速地进行核酸扩增。该技术在1h内能扩增出109靶序列拷贝,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片段的混合物。因其具有特异性强、灵敏度高、快速、准确和操作简便等优点因为其操作简单、快速、特异性高、成本低等优点,成为可以替代PCR的新的核酸扩增技术。本研究采用LAMP技术建立了快速检测普通变形杆菌的反应体系。该方法针对变形杆菌的特异基因atpD,用LAMP引物在线设计引物软件设计引物,在Bst DNA聚合酶作用下60℃恒温反应60min即可完成对变形杆菌DNA扩增过程。扩增产物以2.0%琼脂糖凝胶电泳检测。为确立了最佳反应条件,我们对Mg2+浓度、反应时间、反应温度、BST酶浓度等反应条件进行了优化,建立LAMP反应体系。LAMP反应的总反应体系为25μL,其中FIP和BIP为1.6μmol/L, F3和B3为0.2μmol/L,其它反应组分终浓度分别为:1.6 mmol/L dNTP, 2.5μL Bst酶缓冲液（20 mM Tris-HCl （pH8.8, 25℃）,10 mM KCl,10 mM （NH₄）₂SO₄,2mM MgSO₄, 0.1% .Triton X-100）, 0.4μL （8U） Bst大分子聚合酶,1.5μL目标DNA,纯水补足体积到25μL。将反应混合物在95℃加热5min后放置冰上冷却,在62℃孵育60min,然后在80℃加热10min终止反应。为评价该技术的特异性,我们使用设计的特异引物对11株菌分别进行了LAMP扩增,结果表明:只有变形杆菌为阳性结果,其它菌株均为阴性结果。采用FTA滤膜制备模板进行LAMP反应,其方法的灵敏度分别为6.4×10²CFU/mL。利用LAMP技术直接检测人工污染的肉制品中的变形杆菌,其检出限为3.6×10³CFU/g。实际检测了45份样品,用LAMP方法检测变形杆菌的检出率为88.9%,敏感性为100%,特异性为72.7%,符合率为93.3%,检出时间为1.5h。试验结果表明:本试验所建立的快速检测变形杆菌的LAMP检测方法具有较高的特异性和敏感性,能够满足变形杆菌快速检测的需要。更多还原

【Abstract】 Proteus.vulgaris is a kind of food-borne pathogenic bacteria which is generally distributed in the environment, such as in polluted water, soil and also in intestinal tract of human and animal. It is a conditional pathogenic bacteria, usually polluting the animal-derived food, such as meat and egg. It is leading to gastroenteritis, meningitis, septicemia etc. Traditional method for routine detection of Proteus.vulgaris is complex and time-consuming. It takes from 48 to 72 hours. This method can not fulfil the need for detecting food-borne pathogens rapidly and sensitive. So is need to establish method for detection proteus mirabilis, so as to provide a reference method for sanitary inspection.Loop-mediated isothermal amplification （LAMP） which stands for Loop-mediated Isothermal Amplification is a new nucleic acid amplification method solely developed by Eiken Chemical Co.Ltd in 2000. The LAMP method employs four primers which specifically recognize six distinct sequences on the target DNA and a bst DNA polymerase that has a strand displacemet activity. The target DNA can be amplified with high specificity, efficiency, andrapidity under isothermal conditions ranging from 60 to 65℃.LAMP can amplify a few copies of DNA to 10⁹ in less than an hour. The final products are stem-loop DNA with several inverted repeats of the target and cauliflower-like structures with multiple loops. The LAMP method will be replace PCR because of its simplicity, rapidity, specificity, and cost-effectiveness.Loop-mediated isothermal amplification（LAMP）was applied to quickly detect Proteus DNA. The specific LAMP primers of Proteus.vulgaris were designed on the basis of the published sequence of strain atpD with the LAMP primer design support software program, using primer explorer software. The reaction was carried out at 62℃for 1 hour and the products were separated by 2.0% agarose gel electrophoresis.The reaction conditions were optimized including Mg²⁺ concentration、temperature、time、BST concentration etc. The LAMP reaction was carried out in a 25μLtotal reaction mixture volume with containing 1.6μmol/L each of inner primers FIP and BIP, 0.2μmol/L each of outer primers F3 and B3, 1.6 mmol/L each deoxynucleoside triphosphate, 6.0mmol/L MgSO4, 2.5μL Bst DNA Polymerase Buffer（20 mM Tris-HCl （pH8.8, 25°C）, 10 mM KCl, 10 mM （NH₄）₂SO₄, 2mM MgSO₄, 0.1% Triton X-100）, 0.4μL of Bst DNA polymerase,and 1.5μL of target DNA. The mixture was incubated at 62°C for 60 min in a heating block and then heated at 80°C for 10 min to terminate the reaction.There were 11 bacterial amplified by LAMP method in order to evaluate the specificity of primers. The result of only Proteus was positive and those of other strains were negative. The sensitivities of LAMP assays using the FTA filters as templates were 6.4×10²CFU/mL. The detection limits of LAMP assays obtained from artificially inoculated meet samples were 3.6×10³CFU/g. 45 samples were analyzed The detection rate of LAMP amplification was 91.1%, 100% for sensitivity and specificity of 75.0%, the according rate was 95.6%, the detection time was 2h.The results showed that the LAMP method developed in this experiment was with high specificity and sensitivity, the deveolped LAMP method can meet the demands of Rapid Detection of Proteus.更多还原