利用LAMP法快速检测致病性哈维氏弧菌

Rapid detection of pathogenic Vibrio harveyi by loop-mediated isothermal amplification (LAMP) method

从浙江宁波的发病鲈鱼(Lateolabraxjaponicus)分离到1株致病性哈维氏弧菌(Vibrio harveyi)LY-1。从该菌的DNA样本中成功扩增出大小为873 bp的ToxR基因片段。DNA序列分析表明:该序列与已登录哈维氏弧菌(Vibrio harveyi)ToxR基因同源性为96.57%,与副溶血弧菌(V.parahaemolyticus)、鳗弧菌(V.anguilarum)、创伤弧菌(V.vulnificus)、费氏弧菌(V.fisheri)、溶藻胶弧菌(V.alginolyticus)、霍利斯弧菌(V.hollisae)、拟态弧菌(V.mimicus)、河弧菌(V.fluvialis)的ToxR基因相似性为27.62%～72.49%。选择哈维氏弧菌ToxR基因种内保守区段,设计1套环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术的特异引物,对扩增条件进行优化实验,在65℃孵育45 min的条件下即可完成哈维氏弧菌特异性扩增,成功建立了海水致病菌-哈维氏弧菌的LAMP快速检测方法。结果分析表明:该方法对哈维氏弧菌DNA的最小检出量为1 fg,比PCR法灵敏度高3个数量级。利用LAMP方法快速检测哈维氏弧菌,目前在国内外还未见报道。

A pathogenic Vibrio harveyi strain LY-1 was originally isolated from diseased Lateolabrax japonicus cultured in Ningbo,Zhejiang Province.873 bp-length fragment of ToxR gene cloned from the DNA of V.harveyi strain LY-1 was sequenced and showed that it shared high identity of 99% to those of V.harveyi in GenBank,and had lower similarities to those of V.parahaemolyticus,V.anguilarum,V.vulnificus,V.fisheri,V.alginolyticus,V.hollisae and V.mimicus,with only 27.62%~72.49% identity.A set of loop-mediated isothermal amplification（LAMP） primers were designed and synthesized based on the conservative sections of V.harveyi ToxR gene,and the LAMP for detection of V.harveyi was developed after optimizing the factors.LAMP required only a incubation at 65 ℃ for 45 min,and its detection limit for V.harveyi DNA was 1 fg,which was found to be more sensitive than the commonly used PCR method.To date,this is first report that LAMP method is used to detect V.harveyi rapidly.