DNA染料结合环介导等温扩增技术检测志贺氏菌死活细胞  

Differentiation of the viable and dead cells of Shigella by Loop-mediated isothermal amplificaiton with a DNA binding dye

作　　者：李月[1] 王丽[1] 孙远明[1] 钟青萍[1] 

机构地区：[1]华南农业大学食品学院,广东广州510640

出　　处：《食品工业科技》2011年第8期216-219,共4页Science and Technology of Food Industry

基　　金：广东省农业攻关重点专项(2009A020101004);国家自然科学基金(31000781)

摘　　要：建立了一种DNA染料(EMA)结合环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP)的分析方法(EMA-LAMP),用于有效检测区分病原微生物志贺氏菌的死活细胞。基于志贺氏菌ipaH基因的六个区域设计特异性的引物,检测志贺氏菌的死活细胞。结果表明:浓度为40μg/mL的EMA能够有效抑制109CFU/mL的死细胞扩增,而对相同浓度的活菌扩增没有影响。分析表明,该方法可以有效区分细菌的死活细胞,克服了传统PCR无法区分死活细胞的弊端,同时EMA-LAMP检测方法耗时短,检测灵敏度高,是一种能够有效鉴别病原菌死活细胞的新方法。Conventional DNA-based detection methods can be used to detect both live and dead cells.Since dead cells do not cause disease,which bring a problem in medical or biological studies.The Loop-mediated isothermal amplification（LAMP）method combined with the ethidium monoazide（EMA）treatment was applified with viable,but not dead Shigella cells.LAMP method employs a DNA polymerase and a set of specially designed primers that recognize a total of six distinct sequences on the target ipaH gene conserved in Shigella.The results showed that the concentration of EMA was 40μg/mL,DNA amplification was inhibited derived from dead cell suspensions（1.0×109CFU/mL）,but not inhibit viable cells.These results showed that EMA-LAMP method had a potential for specific detection of viable cells,one hand,EMA had the ability to penetrate selectively through the damaged cytoplasmic membrane of dead cells and to intercalate into DNA,the gene of the dead cells could not be amplified.On the other hand,EMA-LAMP had high sensitivity and specificity.

关 键 词：ETHIDIUM monoazide 环介导等温扩增技术(LAMP) SHIGELLA 死活细胞 

分 类 号：Q789[生物学—分子生物学]