本文選題：食源性致病菌　切入點：LAMP　出處：《中國計量學(xué)院》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：摘要：近年來，國內(nèi)外報道的由食源性致病菌引起的食物中毒事件越來越多。阪崎腸桿菌、單增李斯特菌和志賀氏菌都屬于食源性致病菌。誤食之后引起人類各種疾病，危及生命。為了確保食品的安全性，需要大力加強食品的檢驗力度，建立快速、靈敏的檢測手段。本論文以建立快速檢測這三種致病菌的LAMP方法為主要研究目的，為食品安全的快速檢測提供技術(shù)支持。 本研究針對阪崎腸桿菌16S/23S rDNA IGS序列（NCBI登錄號AY748354.1），單增李斯特菌iap基因（NCBI登錄號NC_003210.1），志賀氏菌ipaH基因（NCBI登錄號HE616529.1）分別設(shè)計LAMP特異性引物，內(nèi)引物FIP/BIP，外引物F3/B3。同樣區(qū)域設(shè)計熒光定量PCR引物，構(gòu)建標(biāo)準(zhǔn)質(zhì)粒。對建立的LAMP反應(yīng)體系進(jìn)行條件優(yōu)化，確定其反應(yīng)體系為：10mmol/L內(nèi)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，雙蒸水補足25μL總體積。在67℃保溫60min。實驗結(jié)果可通過三種方式鑒定，直接肉眼觀察是否有白色沉淀產(chǎn)生或渾濁度變化，添加熒光染料SYBR Green I1μL，在紫外透射儀下觀察是否有綠色熒光反射，同樣也可將反應(yīng)產(chǎn)物凝膠電泳，是否有梯形條帶產(chǎn)生。對設(shè)計的三個菌的LAMP引物做了特異性實驗和靈敏度實驗。實驗結(jié)果顯 示，三組引物特異性良好，靈敏度試驗中梯度稀釋DNA模板，阪崎腸桿菌的檢出限為91fg/uL，單增李斯特菌的檢出限為9.6fg/uL，志賀氏菌的檢出限為27.5fg/uL。在模擬食樣實驗中，分別單獨用單增李斯特菌、阪崎腸桿菌、志賀氏菌、沙門氏菌、金黃色葡萄球菌、大腸桿菌增菌液人工污染脫脂乳，用煮沸法粗提取基因組，用LAMP方法與熒光定量PCR方法做參照檢測特異性，實驗結(jié)果顯示，能準(zhǔn)確檢測出引物所對應(yīng)的菌種，且阪崎腸桿菌LAMP方法的檢出限為10~1CFU/mL，熒光定量PCR的檢出限為10~2CFU/mL；單增李斯特菌LAMP方法的檢出限為3×10~0CFU/mL，熒光定量PCR的檢出限為3×10~1CFU/mL；志賀氏菌LAMP方法的檢出限為4×10~0CFU/mL，熒光定量PCR的檢出限為4×10~1CFU/mL。LAMP檢測方法不僅能達(dá)到熒光定量PCR方法的準(zhǔn)確性，而且檢測限更低，靈敏度更高。 實際樣品檢測樣中，分別與阪崎腸桿菌、單增李斯特菌、志賀氏菌行業(yè)標(biāo)準(zhǔn)檢測法(sN/T1632.1-2005[v6])做比較，結(jié)果表明：76份奶粉中阪崎腸桿菌，LAMP方法的陽性檢出率為3.95%，檢出時間為l.5h。76份奶粉中志賀氏菌，LAMP方法的陽性檢出率為3.95%，檢出時間為l.5h。54件肉制品中，單增李斯特菌的LAMP方法的陽性檢出率為13.0%，檢出時間為2.0h。LAMP方法在對樣品的檢測更加靈敏，且更省時。 本研究分別建立了阪崎腸桿菌、單增李斯特菌和志賀氏菌的LAMP快速檢測方法，開發(fā)單增李斯特菌LAMP快速檢測試劑盒，，建立三種LAMP反應(yīng)結(jié)果目測方法，基本實現(xiàn)了現(xiàn)場快速檢測的目標(biāo)。該方法特異性強，靈敏度高，檢測時間短，為食品中致病菌的檢測提供了一個新的檢測平臺，為檢測機構(gòu)推廣此技術(shù)提供了技術(shù)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.
鏈爺絀跺垎鍒緩绔嬩簡闃磶鑲犳潌鑿

本文編號：1588420