EMA-LAMP方法快速检测鉴别副溶血性弧菌

摘要

A method utilizing DNA dye EMA (ethidium bromide monoazide) binding with LAMP (loop-mediated I-sothermal amplification) for Vibrio parahaemolyticus ( Vp) discrimination in pure culture containing a mixture of viable and/or dead cells was established. A set of four primers and two loop primers were designed aiming at six sites in thermolabite hemolysin (tlh) gene specific sequence of Vp to carry out the detection. The result showed that concentration at 8.0μg/mL of EMA or above could effectively inhibit the amplification of DNA derived from dead cells of Vp with cell suspension of 1×108 cfu/mL under the light exposure for at least 25 min, whereas the same concentration of EMA to treat viable cells of Vp had no effect on the amplification. After treated with EMA, the minimum level was 1. 0×102 cfu/mL to detect viable cell in a mixed liquid containing different proportion of viable and/or dead Vp cells. Therefore, the EMA-LAMP method was more effective method to discriminate the viable cells from the dead ones than EMA-PCR; it is a rapid, sensitive and more efficient new method to discriminate the viable cells from the dead cells of Vp.%建立将DNA染料EMA(ethidium bromide monoazide)结合环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)的方法(EMA-LAMP),用于检测鉴别副溶血性弧菌(Vibrio parahaemolyticus)死/活菌细胞.针对副溶血性弧菌不耐热溶血素基因tlh(thermolabile hemolysin)特异性序列的6个位点设计4条引物及2条环引物,进行检测.结果表明,浓度为8.0μg/mL或更高浓度的EMA,至少经25 min的曝光处理,能够有效抑制浓度为1 x 1o8 cfu/mL的副溶血性孤菌死细胞的扩增,而对用相同浓度EMA处理的副溶血性弧菌活细胞扩增没有影响.经EMA处理,含有不同比例的副溶血孤菌死细胞和活细胞的混合液中,活菌的最小检测限为1.0×102 cfu/mL.EMA-LAMP方法比EMA-PCR方法区分死活细胞中的活细胞更为有效,是一种能够快速、灵敏且更为有效鉴别副溶血性弧菌死活细胞的新方法.