【摘要】 目的建立伤寒沙门氏菌AFLP—银染体系。方法对EcoRⅠ和MseⅠ两种限制性内切酶用量、酶切时间、连接产物及预扩增产物的稀释倍数等进行研究。结果建立了伤寒沙门氏菌AFLP—银染体系在20μl的酶切体系中,含基因组DNA 250ng,EcoRⅠ和MseⅠ各2.5U,37℃下酶切3h;21℃连接过夜;连接产物稀释10倍后进行预扩增;预扩增产物稀释50倍后再进行选择性扩增;选择性扩增完成后加等量Loading buffer,90℃变性3min,立即冰浴;在6%变性聚丙烯酰胺凝胶上进行电泳分析;银染法检测PCR产物。结论AFLP—银染体系有望在伤寒沙门氏菌的多态性研究和流行病学调查中发挥重要作用。更多还原

【Abstract】 Objective To establish the AFLP Silver—staining Analysis System of Salmonella Typhi. Methods Study on the quantity of EcoRⅠand MseⅠapplied, the time of the restriction of endonuclease digestion, the dilution times of the ligation mixture and the pre-amplified product. Results Results AFLP silver—staining analysis system of Salmonella Typhi was established. The purified Salmonella Typhi genomic DNA 250ng, EcoRⅠ2.5U and MseⅠ2.5U were in a reaction volume of 20μl, and incubated at 37℃ for 3 h. Adaptors were ligated at 21℃ overnight. 1∶10 dilution of the ligation mixture was used as templates for pre-amplification. 1∶50 dilution of the pre-amplified product was used as templates for selective amplification. At the end of the selective amplification, the Salmonella Typhi samples were denatured by adding an equal volume of loading buffer and heated up at 90℃ for 3 min, then cooled on ice immediately. The PCR reaction products were analysed on 6% denaturing acrylamide gels and then stained by silver. Conclusion Conclusion AFLP could be a powerful tool in polymorphism study and molecular epidemiology investigation of Salmonella Typhi.更多还原