Development Of Real-time Loop Mediated Isothermal Amplification System For Detection Of Three Kinds Of Foodborne Pathogens

In recent years,the world's food safety have been a hot topic in the world.Among them,the incidence of bacterial foodborne diseases,which is one of the most widely distributed and most common diseases,is also on the rise.Conventional testing techniques have their own advantages,however,these technologies are only carried out in the laboratory,and have many inconveniences such as long detection period and complicated operation,they are not suitable for on-site testing.Loop-mediated isothermal amplification(LAMP),that amplifies DNA with high specificity,efficiency and rapidity under isothermal conditions.This method employs a Bst DNA polymerase and a set of four specially designed primers that recognize a total of six distinct sequences on the target DNA.This study established a rapid detection technique for three common foodborne pathogens: Campylobacter jejuni,Enterococcus faecalis and Staphylococcus aureus,and combined it with a portable on-site test box to achieve the Rapid on-site detection of three food-borne pathogens.Establish a real-time fluorescent Loopmediated isothermal amplification(LAMP)screening system for simultaneous detection of campylobacter jejuni,enterococcus faecalis and staphylococcus aureus enabling on-site highthroughput detection of pathogens,targeting hipO gene,EF0027 gene and nuc gene respectively.Reaction conditions of real-time LAMP were improved for efficient primer sets,such as teperature and primer ratio.The specificity and the detection limit of real-time LAMP was evaluated,which were compared with PCR Simultaneously.LAMP was carried out in a total 25?L reaction mixture containing 1.6 ?M each FIP and BIP,0.2 ?M each F3 and B3,Warm Start LAMP 2×Master Mix 12.5?L(New England Biolabs),50× fluorescent dye 0.5?L,1?L double-stranded target DNA,8.5?L ddH2 O,followed by incubation at 65? for 1h and heating at 80? for 5min to terminate the reation.In the present study,three real-time LAMP assays were established for rapid,sensitive,and specific detection of campylobacter jejuni,enterococcus faecalis and staphylococcus aureus.The results indicated that the LAMP assays had similar analytical performance compared to convential PCR.The LAMP assays of the three bacteria has high detection specificity,and the detection sensitivity is higher than the PCR method at least 10 times.The detection limit of the real-time fluorescence LAMP detection method of Campylobacter jejuni,Enterococcus faecalis and Staphylococcus aureus is 34.2 fg/?L,98 fg/?L,1.08 fg/?L respectively.The real-time LAMP assay has its advantages in some areas such as simple equipment,low cost,simple process and short time.Therefore,it's a portable and user-friendly assay for rapid detection of foodborn bacteria on-site.