三种食源性致病菌的LAMP检测方法研究

发布时间：2018-03-09 11:53

  本文选题：食源性致病菌　切入点：LAMP　出处：《中国计量学院》2012年硕士论文　论文类型：学位论文

【摘要】：摘要：近年来，国内外报道的由食源性致病菌引起的食物中毒事件越来越多。阪崎肠杆菌、单增李斯特菌和志贺氏菌都属于食源性致病菌。误食之后引起人类各种疾病，危及生命。为了确保食品的安全性，需要大力加强食品的检验力度，建立快速、灵敏的检测手段。本论文以建立快速检测这三种致病菌的LAMP方法为主要研究目的，为食品安全的快速检测提供技术支持。 本研究针对阪崎肠杆菌16S/23S rDNA IGS序列（NCBI登录号AY748354.1），单增李斯特菌iap基因（NCBI登录号NC_003210.1），志贺氏菌ipaH基因（NCBI登录号HE616529.1）分别设计LAMP特异性引物，内引物FIP/BIP，外引物F3/B3。同样区域设计荧光定量PCR引物，构建标准质粒。对建立的LAMP反应体系进行条件优化，确定其反应体系为：10mmol/L内引物（FIPBIP），10mmol/L外引物（F3B3），10mmol/LdNTPs，5M Betaine，16mmol/L MgSO_4，20mmol/LKCl，20mmol/L (NH_4)_2SO_4，0.2%TritonX-100，8U Bst DNA Polymerase，1μL DNA模板，双蒸水补足25μL总体积。在67℃保温60min。实验结果可通过三种方式鉴定，直接肉眼观察是否有白色沉淀产生或浑浊度变化，添加荧光染料SYBR Green I1μL，在紫外透射仪下观察是否有绿色荧光反射，同样也可将反应产物凝胶电泳，是否有梯形条带产生。对设计的三个菌的LAMP引物做了特异性实验和灵敏度实验。实验结果显 示，三组引物特异性良好，灵敏度试验中梯度稀释DNA模板，阪崎肠杆菌的检出限为91fg/uL，单增李斯特菌的检出限为9.6fg/uL，志贺氏菌的检出限为27.5fg/uL。在模拟食样实验中，分别单独用单增李斯特菌、阪崎肠杆菌、志贺氏菌、沙门氏菌、金黄色葡萄球菌、大肠杆菌增菌液人工污染脱脂乳，用煮沸法粗提取基因组，用LAMP方法与荧光定量PCR方法做参照检测特异性，实验结果显示，能准确检测出引物所对应的菌种，且阪崎肠杆菌LAMP方法的检出限为10~1CFU/mL，荧光定量PCR的检出限为10~2CFU/mL；单增李斯特菌LAMP方法的检出限为3×10~0CFU/mL，荧光定量PCR的检出限为3×10~1CFU/mL；志贺氏菌LAMP方法的检出限为4×10~0CFU/mL，荧光定量PCR的检出限为4×10~1CFU/mL。LAMP检测方法不仅能达到荧光定量PCR方法的准确性，而且检测限更低，灵敏度更高。 实际样品检测样中，分别与阪崎肠杆菌、单增李斯特菌、志贺氏菌行业标准检测法(sN/T1632.1-2005[v6])做比较，结果表明：76份奶粉中阪崎肠杆菌，LAMP方法的阳性检出率为3.95%，检出时间为l.5h。76份奶粉中志贺氏菌，LAMP方法的阳性检出率为3.95%，检出时间为l.5h。54件肉制品中，单增李斯特菌的LAMP方法的阳性检出率为13.0%，检出时间为2.0h。LAMP方法在对样品的检测更加灵敏，且更省时。 本研究分别建立了阪崎肠杆菌、单增李斯特菌和志贺氏菌的LAMP快速检测方法，开发单增李斯特菌LAMP快速检测试剂盒，，建立三种LAMP反应结果目测方法，基本实现了现场快速检测的目标。该方法特异性强，灵敏度高，检测时间短，为食品中致病菌的检测提供了一个新的检测平台，为检测机构推广此技术提供了技术支持。
[Abstract]:Abstract: in recent years, more and more events reported at home and abroad of the poisoning caused by pathogenic bacteria food. Enterobacter sakazakii, Listeria bacteria Lester and Shigella are foodborne pathogens. After eating caused by a variety of human diseases, endanger life. In order to ensure food safety, we need to strengthen food inspection efforts to establish a rapid and sensitive detecting methods. In this paper, the establishment of rapid detection of three kinds of pathogenic bacteria LAMP method as the main research objective, rapid detection for food safety to provide technical support.
In this study, 16S/23S rDNA of Enterobacter sakazakii IGS sequence (NCBI accession number AY748354.1), Listeria bacteria Lester IAP gene (NCBI accession number NC_003210.1), Shigella ipaH gene (NCBI accession No. HE616529.1) respectively design LAMP specific primers and inner primers FIP/BIP, F3/B3. also introduced regional design of fluorescent quantitative PCR primer construction of standard plasmid., to optimize the LAMP reaction system is established, the reaction system is 10mmol/L (FIPBIP), inner primer 10mmol/L primers (F3B3), 10mmol/LdNTPs, 5M, Betaine, 16mmol/L, MgSO_4,20mmol/LKCl, 20mmol/L (NH_4) _2SO_4,0.2%TritonX-100,8U Bst DNA Polymerase, 1 L DNA template, double distilled water is made up of 25 mu L the total volume at 67 DEG C for 60min.. The experimental results can be identified by three ways, direct visual observation whether a white precipitate produced or turbidity change, add SYBR Green I1 fluorescent dye L in purple Whether the green fluorescence reflex was observed under the external transmission instrument, the reaction product gel electrophoresis and trapezoidal strip production could also be produced. Specific experiments and sensitivity tests for the three primers of the LAMP were designed.
In the three sets of primers with good specificity, sensitivity test in gradient dilution DNA template, the detection of Enterobacter sakazakii detection limit is 91fg/uL, Listeria bacteria Lester limit is 9.6fg/uL, the detection limit of 27.5fg/uL. in food samples in the experiment simulation of Shigella, separately with Listeria bacteria Lester, Enterobacter sakazakii, Shigella, Salmonella, Staphylococcus aureus, Escherichia coli bacteria artificially contaminated skim milk by boiling method extract genome, using LAMP method and fluorescent quantitative PCR method for detection of specific reference, the experimental results show that can accurately detect the corresponding primers and methods of Enterobacter sakazakii strains, LAMP coli detection limit is 10~1CFU/mL, the detection limit of 10~2CFU/mL fluorescence quantitative PCR detection method; Lester strain LAMP Listeria detection limit is 3 * 10~0CFU/mL, fluorescence quantitative PCR limit is 3 * 10~1CFU/mL; the detection limit of Shigella LAMP method was 4 The detection limit of fluorescence quantitative PCR is 4 * 10~1CFU/mL.LAMP by X 10~0CFU/mL. The detection method not only achieves the accuracy of fluorescence quantitative PCR, but also has lower detection limit and higher sensitivity.
The actual sample testing samples respectively, and Enterobacter sakazakii, Listeria bacteria Lester, Shigella industry standard detection method (sN/T1632.1-2005[v6]) for comparison, the results showed that: 76 milk powder samples of Enterobacter sakazakii in the LAMP method, the positive rate was 3.95%, the detection time is l.5h.76 in milk powder of Shigella, LAMP methods the positive detection rate was 3.95%, the detection time of l.5h.54 pieces of meat products, LAMP method of Listeria bacteria Lester the positive detection rate was 13%, the detection time of 2.0h.LAMP method in the detection of samples is more sensitive and more time-saving.
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