一种改良型检测群体感应信号分子的TLC方法

A modified TLC method for detecting quorum sensing signal molecules of N-acyl-homoserine lactones in bacteria

微生物群体感应信号主要包括三类(AI-1,AI-2和AI-3),其中以AI-1(即N-酰基高丝氨酸内酯,Acyl-homoserine lactones,AHLs)的研究最为广泛。以往针对AHLs的检测主要是传统的薄层色谱法(Thin Layer chromatograph,TLC),即采用报告菌株、显色剂X-gal以及半固体培养基三者混合后平铺于TLC板上,通过生物显色反应来进行定性判断。但这一方法在实际操作中存在一些不足,包括显色不均匀、蓝色斑点发散、以及分辨率不高等问题,因此该方法还具有很大的改进空间。利用根癌农杆菌(Agrobacterium tumefaciens)A136(pCF218)建立了一种改良型的TLC方法,主要包括设计一套简易型的制胶板、改变铺板程序以及节省X-gal用量等。结果表明,改良后的方法使得显色斑点更聚焦,显色强度和线性迁移率效果更好,检测分辨率更高,更适合混合型样本。同时该方法操作简便、无需专业培训、初学者易上手,增加了方法的普适性和有效性。

Quorum sensing(QS)is the unique,diverse group behavior phenomenon occurred when the bacterium grows to a certain density and responses mutually,and it is responsible for the gene expression and the regulation.There are three kinds of QS singanls,including AI-1,AI-2 and AI-3.Among them,AI-1,i.e.N-Acyl-homoserine lactones(AHL),is the most well-known QS signals and regulates the expression of many physiological characteristic in Gram negative bacteria.The traditional dection method main depends on thin-layer chromatography(TCL).However,some shortcomings still exist in this classical methods,including uneven pigment,color-spot disperse,and low resolution.So,it is important to set up an optimizatd method for rapid,simple and effective detection of AHLs.In this study,a modified protocol was developed based on the traditional TLC method,including design a preparating-agar plate,modified the operating procedure to combine the TLC with biosensor,and save the X-gal′usage.The results showed that the new protocal have higher resolution,stronger pigmentation,and better surface mobility.More interestingly,the modified method can bring convenient for the natural samples or mixed samples.This new method is efficient,sensitive,inexpensive and easy to apply for the detection of AHLs and analogue.