| Nowadays,with such advanced technology,people have caused serious pollution to the environment while raising our living standards,including the random disposal of daily output garbage,industrial "three wastes",pesticide residues and water pollution.Resulting in a series of environmental problems seriously threaten human survival and health.Therefore,in order to prevent it from happening,researchers have researched and developed many detection methods that can detect trace toxic substances in the environment.Among them,chromatographic and mass spectrometry techniques used in physical chemistry can effectively detect traces in the environment.However,due to its shortcomings such as complicated operation process,high cost and long test time,it has not been widely used in the detection of environmental poisons.Subsequently,people have developed microbial detection systems.Among them,the Ames test and SOS/umu test for short-term detection of genotoxic substances have been widely used because of their advantages such as short detection period,low test sample consumption,and high reliability.In this study,based on the SOS response principle,five SOS promoters recA,imuA,qnrB,sulA and cda were selected and the bacteriophage cleavage gene SRRz was selected as the downstream expression gene of these promoters.Five engineering bacteria were constructed using E.coli BL21 as the host strain.Then,six typical genotoxic substances were incubated with 5 recombinant lytic strains at 37 ℃,respectively.After about 0.5-1 h,the bacterial density began to decline and the change of bacterial turbidity was observed with the naked eye,which verified the feasibility of the five detection systems for the detection of genotoxic substances and the advantages of short detection time,easy operation,and low cost.Hereafter using the dose-effect relationship between the cell death rate of drug-induced recombinant strains and drug concentration,the detection limits of 5 recombinant strains for different chemicals are calculated.The results showed that the recombinant lytic strain E.coli BL21 p UC-RST(rec A promoter)showed a stronger response to the four chemicals 5-fluorouracil,cumylhydroperoxide,dichlofluanid and dimethylsulfate,with detection limits of 1.2、2.1、1.1and 6.6 μg/m L,compared with the traditional SOS/umu experiment,the LOD value decreasedby 92.5%,58.0%,66.7% and 82.6%.In addition,the recombinant strain E.coli BL21 pUC-SST(sul A promoter)also showed very good sensitivity to dichlofluanid and dimethylsulfate,compared with the traditional SOS/umu experiment,the LOD value decreased by 72.7% and 92.1%,however,the other three strains showed different results for different drugs.In this study,screening for the genotoxicity of 40 commercially available pesticides using the engineered strain E.coli BL21 pUC-RST with better sensitivity showed that 16 of the pesticides were genotoxic.And compared their detection limits with the maximum residue limits of pesticides specified in the national standard GB 2763-2016 "Maximum Residue Limits of Pesticides in Food".The results showed that the recombinant lytic strain constructed in this study has a good response to the commercially available pesticide products,and can detect pesticide contents below the national maximum residue limit.Therefore,the recombinant cracking screening system constructed in this study can be applied to the detection experiments of pesticide residues in the environment and has certain practical value.In summary,the genotoxic substance detection system constructed in this study has the following advantages: easy to operate,short detection period,low sample consumption and low cost;and it has a good response to genotoxic substances,high sensitivity,and a wide detection range,which can be applied to the on-site detection of actual environmental samples and achieve high-throughput screening. |
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