Development Of The Methods For Quick Detection Of E.coli And Pseudomonas Aerugunosa

The harmful microorganisms in enviorment,water and food arenon-negligible threat to human health. In recent years, The diseases causedby foodborne pathogens mainly including Escherichia coli（E.coli）, Vibrioparahaemolyticus, Listeriosis, Golden staphylococcus, Salmonella, etchave occurred frequently worldwide. Millions of people died frommicroorganism-contaminated food and water all over the world. Theidentification of foodborne-pathogens is the key point for the preventionand control of foodborne disease, and the effective way for protecting foodsecurity as well. To effectively ensure food safety,it is urgent to developsome simple, rapid, efficient measures for detecting foodborne pathogens.In this study, an amperometric biosensor and a photoelectric biosensorwere established for the detection of Escherichia coli（E.coli）,Pseudomonas aeruginosa（P. aeruginosa）, and the effects of the methodswere evaluated. The research process of the sensor methods and thedetecting results were summarized as follows:1. The development of photoelectric sensor method for detection of P.aeruginosa: Based on the mechanism that P.aeruginosa secretescytochrome oxidase catalyzing cytochrome C which oxidizeshydrochloride dimethyl-phenylenediamine and produces a colorreaction.There is a significant linear relationship between the color degreeof the reacton and the concentration of P aeruginosa. According the colorreaction, a portable optical test strip was developed, in which the reagentof hydrochloride dimethyl-phenylenediamine were coated. After the teststrip was inserted into photoelectrical sensor, samples were added to thereaction area of the strip.Then the light reflection was recorded as the sensor singal. A standard calibration curve was established between thelight reflection and the concentrations of P aeruginosa. The results showedthat the equation of the method for detecting P aeruginosa isY=1527.35714-133.96429X, R=-0.99974, the detectable limit is103CFU/mL, and the time for detecting a sample is less than5min that isquicker than other methods.2. The development of photoelectric sensor method for detection ofE.coli： E.coli secretes β-galactosidase under the inducement ofisopropyl-β-D-thio-galactopyranoside （IPTG）, β-galacosidase catalyzesChlorophenol Red-β-D-galactopyranoside （CPRG） to Chlorophenol red（CPR） along with a color reaction from yellow to red. The color change ofthe system has a good correlation with the concentration of E.coli. Thelight reflection of the color reaction are recorded with the photoelectricsensor by adding the reaction reagents to the photoelectric type test stripwe made. According to the inverse relationship between the concentrationof E.coli and light reflection, a method of photoelectric sensor fordetecting E. coli was developed. The results showed that the optimum pHvalue for the reaction system is7.5and the time for the enzymatic reactionis4hours. The logarithm value of the bacteria concentration in samplespresented a significant correlation with the reflected light signal value（r=-0.98944,p<0.01）,the linear equation is Y=1426.10485-97.23368X, thedetection limit is104CFU/mL, the time for detecting a sample is less than5min.3. The development of amperometric sensor method for detection ofE.coli:The Secretion of β-glycosidase from E.coli is induced byIsopropyl-β-D-thio-galactoside （IPTG）. This enzyme can specificallycatalyze p-Aminophenyl-β-D-galactopyranoside （PAPG） top-Aminophenol （PAP）, a substance with significant electrochemicalactivity. The value of current produced by the redox reaction shows alinear correspondence relationship with the concentration of E.coli. Anamperometric sensor with disposable screen printed electrode （SPE） was used to detect the current, the standard curve between the electrochemicalsignal of PAP and the concentration of E.Coli. was plotted and the reactionconditions was optimized.The results showed that the peak current of thePAP presents at200mV. The optimized pH value and enzymatic reactiontime for this sensing system are7.5and45min, respectively. Bacterialdensity in the sample indicates satisfactory correlation with the peakcurrent and the linear equation is y=1.1346×10^-6+1.8950×10^-5x，R²=0.98943（p<0.01）with the detection limit of103CFU/mL,and detecting asample spends not more than10min.In conclusion, the three sensor methods for detecting P.aeruginosaand E. coli in food,water and environment samples are quick, economical,convenient,simple and easy to perform.After further improvement andoptimization, hopefully it can be applied in the practice for fast screeningof mass samples and provides a new way for the identification of harmfulmicroorganisms.