沙门氏菌属随机扩增多态性DNA PCR条件的优化  

Optimization of RAPD PCR Assay for Salmonella Detection

作　　者：粟婉媛[1] 侯家奎[2] 柳婷[1] 郭爱玲[1] 王洪江[1] 谢跻[1] 马美湖[1] 

机构地区：[1]华中农业大学食品科学技术学院,湖北武汉430070 [2]许昌职业技术学院,河南许昌461000

出　　处：《食品科学》2009年第20期320-323,共4页Food Science

基　　金：科技部农学教育学实验用微生物资源整合项目(2005DKA21208-6;2006DKA21208-6)

摘　　要：目的:以5′-CCGAAGCTGC-3′为引物,利用随机引物扩增多态性DNA技术(random amplified polymorphic DNA,RAPD)对沙门氏菌属的几种菌株进行扩增,以确定RAPD的最终优化条件。方法:首先选用25μl体系PCR和50μl体系PCR做对比,以确定进行RAPD实验最佳的PCR体系。其次进行PCR反应体系条件的优化,对PCR复性温度、引物浓度、Mg2+浓度进行条件优化。最后对初步确定的优化结果进行不同琼脂糖胶浓度梯度的优化以确定RAPD的最终优化条件。结果:RAPD优化条件为PCR复性32℃,25mmol/LMg2+溶液体积2.0μl,10μmol/L引物体积0.2μl,琼脂糖胶浓度1.0%。结论:在优化条件下,以引物5′-CCGAAGCTGC-3′对沙门氏菌属的3种标准菌株,9株食品分离株进行扩增,得到的电泳条带最多,且最清晰。A random amplification of polymorphic DNA （RAPD）-PCR assay was developed for detecting Salmonella using a primer of 5＇ -CCGAAGCTGC-3＇. In order to obtained distinctive and reliable results, the optimal PCR reaction conditions （namely PCR system volume, renaturation temperature, primer concentration and Mg^2＋ concentration） and agarose concentration for electrophoresis were investigated. Results showed that the optimal RAPD-PCR reaction was performed in a 25μ I system containing 2.0μl of 25 mmol/L MgCh and 0.2μl of primer at 32 ℃ and the optimal agarose concentration was 1.0%. Under such conditions, sufficient and clear electrophoresis strips were observed in RAPD-PCR detection of 3 typical Salmonella strains and 9 isolated Salmonella strains.

关 键 词：沙门氏菌 地方菌株 随机扩增多态性DNA 优化 

分 类 号：TS201.3[轻工技术与工程—食品科学]