A Real-time Turbidimeter-based Loop-mediated Isothermal Amplification Assay For Rapid Detection Of E.coli O157

With its higher and higher incidence, Enterohemorrhagic Escherichia coli (EHEC)O157had been listed as an new food-borne pathogens by the world health organization. Ithad become the top priority to develop a detection method of EHEC O157with highefficiency and specificity due to its long time consuming, complex steps and loss ofmanpower as well as material resources by traditional detection methods. To study thenucleic acid structure and molecular features of EHEC O157on the level of molecularbiology and develop the loop-mediated isothermal amplification (LAMP) will probablyreplace the PCR detection and become a new direction for rapid detection.LAMP real-time turbidity method was to use the turbidity instrument monitoring theisothermal amplification process. It was mainly through a by-product of white precipitatemagnesium pyrophosphate in the process of amplification reaction which was proportionalto the expansion amount of target genes in the reaction liquid. In other words, it judged thedegree of reaction by whether the precipitation was produced as well as the starting time.In this research, the specific primers of the LAMP were designed aimed at the antigengene rfbE and virulence genes stx1and stx2which produce Vero toxin EHEC O157, andthe reaction conditions were confirmed and optimized. The most suitable reaction systemcontains a1.6μM concentration of each inner primer (FIP and BIP), a0.2μMconcentration of each outer primer (F3and B3),20mM Tris-HCl（pH8.8）、10mM KCl、8mM MgSO4、10mM （NH4）2SO4、0.1％Tween20、0.8M BETAINE、8mM MgSO4、1.4mM dNTP、8U of the Bst DNA Polymerase and2μl DNA. The mixture was incubatedat63℃for60min and then heated at80℃for5min to terminate the reaction.The specificity, stability and sensitivity of the3primers were evaluated with theoptimized system of performing the LAMP method. The results showed that the lowestdetection limit of new method was103CFU/mL after optimized, which was100timesmore sensitive than the PCR method, and the minimal adding quantity of100CFU/25g(mL) could be detected. This method had been used to detect200food samplesin Zhuhai city, which provided basic data for the food risk assessment.LAMP real-time turbidity method solved the problem that the reaction conditionscould only be optimized by the reaction end point in the establishment of the regularLAMP method, and the new method was not need to analyze the reaction products by gelelectrophoresi, so it could get accurate and reliable data with high-efficiency. The new method had the advantages of rapidness, sensitivity and easy operation, so it is a goodmethod for fast screening food-borne pathogens.