Predictive Modelling Of Escherichia Coli O157:H7by Real-time PCR Method

Escherichia coli O157:H7(E.coli O157:H7) is one of the most common foodborne pathogen in food. This bacterium has strong pathogenicity along with a high death rate, which should be paid high attention around the world. Currently, the use of predictive microbiology models in food to describe and predict dynamic changes of microorganisms has become one of the effective measures to monitor microorganisms. Food predictive microbiology models can make quick assessments and real-time monitoring of the quality and safety of food without the traditional plate culture method that count numbers of microorganisms. Model database established now mainly includes ComBase, PMP, and FSP. The basic data of these models is obtained by traditional plate culture method which can be more complex and time consuming; they are also lack in specificity and sensitivity. In contrast, Real-time PCR is favored by many domestic and foreign scholars for its characteristics like strong specificity, high sensitivity, simple operation and accurate quantification. At the moment, the reports of predictive microbiology models established by Real-time PCR method are scarce. Therefore, this paper selected E.coli O157:H7as the research object. Firstly we established the rapid detection technology of E.coli O157:H7by Real-time PCR method, then applied this method to establish the growth model of E.coli O157:H7under different temperatures. Compared with the models based on the traditional plate culture method, we could analysis the feasibility and reliability of Real-time PCR method to establish growth models. The specific contents and results are as follows:1、We established rapid detection method of E.coli O157:H7based on DNA by Real-time PCR and evaluate the feasibility of this method by parameters such as specificity, sensitivity and quantitative linear range. Results showed that this method had strong specificity. We choose the specific gene primers rfbE of E.coli O157:H7to detect the common spoilage bacteria and pathogens in meat and no amplification plot were seen except E.coli O157:H7; The sensitivity detection of E.coli O157:H7can reach to101cfu/mL. This Real-time PCR method is simple and convenient for the detection of samples which can be finished within6h. No difference was observed in quantitative detection between Real-time PCR and the traditional plate culture method. Thus, the Real-time PCR method based on DNA can make quantitative detection of E.coli O157:H7rapidly and accurately. 2、Based on the results above, we used Real-time PCR method to establish the primary growth models of E.coli0157:1H under different temperatures(10、15、20、25.30℃), including the modified Gompertz model and Logistic model. The two models could both fit the growth curve of E.coli O157:H7under different temperatures by Real-time PCR method and traditional plate culture method. Every curve showed obvious lag phase, logarithmic phase and stable phase, and the temperature had a significant impact on the growth rate and lag phase of microorganisms. The growth rate increased obviously and the period of lag phase shortens quickly as the temperature was higher. Through correlation coefficient (R2), root mean square error (MSE), bias factor (Bf) and accuracy factor (Af), we assessed the accuracy of the models. The values of R2were generally above0.99and MSE were less than0.1log cfu/mL which showed that the fitting degree of models is appropriate. The Bf values and the Af values are all in the acceptable scope, so the models have higher accuracy. Compared with the models based on traditional plate culture method, the models established on Real time PCR method had no significant difference. These models can be used to predict the growth of E. coli O157:H7and solve the problems of traditional predictive models such as labor-intensive and time-consuming.