关键词: 细菌, 酯酶, 代谢活性, 噻唑橙荧光染料, PCR, DNA 扩增, 荧光

Abstract:

In order to develop a new efficient technology for detecting the bacteria alive,a novel fluorescent dye,namely,thiazole orange monoazide ( TOMA),was designed and synthesized based on the metabolic activity of esterasein bacteria,and the molecular structure of TOMA was determined by means of 1H NMR and HRMS. Moreover,the properties of TOMA were preliminarily validated in terms of the intact membrane penetration,the hydrolysisof esterase and the inhibition on the DNA amplification in the PCR.The results show that ( 1) after the treatmentwith 10mg /L TOMA at the room temperature for 10min,the living cells of E.coli O157: H7 emits strong fluorescence,which indicates that TOMA effectively penetrates the intact cell membrane and it possess a good capabilityto bind DNA; ( 2) the hydrolysis rate of TOMA treated by the immobilized lipase is up to 83%，which meansthat the ester bond can be broken by the relevant enzymes; and ( 3) when TOMA is applied to the real-time fluorescentquantitative PCR,the molecular shows an obvious inhibition effect on the DNA amplification.It is thus concludedthat the molecular design of TOMA achieves its desired results.

Key words: bacteria, esterase, metabolic activity, novel thiazole orange fluorescent dye, PCR, DNA amplifica-tion, fluorescence