Study On A Loop Mediated Isothermal Amplification (LAMP) For The Detection Of Escherichia Coli O₁₅₇ In Beef

Escherichia coli O157: H7 was firstly reported by T.Escherich in 1885 and listed as a major food-borne pathogen which can cause diarrhea. Most food-borne intoxication affairs drived from beef and plain milk, and less than 100 individual bacteria can result in intoxication. At present time, the main methods for isolation and identification of E. coli O157: H7 include routine cultivation method, immunologic test and PCR. Traditional method for routine detection of E. coli O157: H7 is complex, time-consuming and lower sensitivity. The immunologic test had low sensitivity. The PCR methods provide powerful tools for rapid, specific, and sensitive detection of food-borne pathogens. However, owing to the expensive systems required and complicate electrophoretic analysis, this application is still not very common in laboratories. A loop-mediated isothermal amplification (LAMP) technology, which can provide a sensitive, specific, simple and cost-effective test for the rapid detection of E. coli O157: H7, was conducted in this assay. It can popularize and promote in common laboratories.This research used the rfbE gene sequence of E. coli O157: H7 (Genbank S83460) as target sequences with the help of Primer Explorer software (http://primerexplorer.jp/e/index.html) for LAMP primers design. The reaction conditions were optimized including the amount of Bst DNA polymerase, dNTPs and Mg2+ concentration etc. The LAMP mixture was made in 25μL of reaction mixture containing a 1.6μM concentration of each inner primer (FIP and BIP), a 0.2μM concentration of each outer primer (F3 and B3), 0.6 mM of each dNTP, 3.0 mM MgCl₂, 10×Bst DNA polymerase reaction buffer, 8 U of the Bst DNA polymerase large fragment, 1μL of isolated DNA templates and sterilized double-distilled water. The mixture was incubated at 64℃for 20 min and then heated at 80℃for 10 min to terminate the reaction. The positive reaction can be determined by the observable presence of the white precipitate in the reaction mixture.For an assessment of the sensitivity and detection limit of artificial contamination of LAMP, PCR with the two outer primers (F3 and B3) of LAMP was performed. We extracted DNA using the commercial kit. The detection limit of pure bacterial culture was 9.8 CFU/mL and the detection limit of artificially contaminated chicken sample was 68 CFU/g with LAMP detection for about two hours. In contrast, the detection limit of pure bacterial culture was 980 CFU/mL and the detection limit of artificially contaminated beef sample was 6.8×10³ CFU/g with PCR detection for about three hours. Our results showed this LAMP assay had a high specificity and time-saving for the detection of E. coli O157: H7 by amplifying a fragment of rfbE of three E. coli O157 strains while other sixteen non- E. coli O157 strains.The effects of three methods of DNA extraction from E. coli O157: H7 in beef sample on detection of E. coli O157: H7 were compared. The results showed that LAMP had less requirements for performing a successful detection.The result indicated that LAMP had the potential to replace PCR because of its simplicity, rapidity, specificity and cost-effectiveness. In conclusion, we developed a new and rapid molecular biological method for specific detection of E. coli O157: H7 in food sample as a basis of a technology platform.