Rapid Detection Of Shigella Using Loop-Mediated Isothermal Amplification

Shigella is one of the wide spread baeterial pathogens mostly exist in food which are rich in protein such as raw meat, dairy products and so on. The method of Shigella detecting now is traditional which has many shortcomings such as complicated operation, long-time detecting which costs 3～5 d. Loop-Mediated Isothermal Amplification (LAMP), a new method for detecting Shigella established in this paper, has more advantages than traditional method in time-cost and sensitiveness.FTA filters are a fibrous matrix impregnated with chelators and denaturants that trap and lyse microorganisms upon contact with filter-based technology, templates from a low numbers of target cells can be prepared efficiently and rapidly with minimal handling and sample loss. In this research, we use the FTA filters for extracting DNA with the LAMP method to detect shigella.The specific LAMP primers of shigella were designed on the basis of the published sequence of ipaH gene with the LAMP primer design software online-program. The concentration of primer, Mg2+ and Bst DNA polymerase are majorized to established the best system.The LAMP reaction was carried out in a 25μL total reaction mixture volume with containing 0.8 mM each of inner primers FIP and BIP, 0.2 mM each of outer primers F3 and B3, 0.3 mM each deoxynucleoside triphosphate, 0.3 mM MgCl₂, 2.5μL ThermoPol buffer (20 mM Tris-HCl (pH8.8, 25°C),10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgSO₄, 0.1%, Triton X-100), 0.5μL 8 U of Bst DNA polymerase,and1μL the specified amounts of target DNA. The mixture was incubated at 62°C for 60 min in a heating block and then heated at 80°C for 2 min to terminate the reaction.Rapid detection of the LAMP method which compared with the PCR detection methods were evaluated. Under the optimum reaction conditions, a rapid technique for detection of shigella was initially established by LAMP. The LAMP reaction can produce a large number of visible precipitation of magnesium pyrophosphate in process.The LAMP assay detection limit was detecting 62 CFU/mL of pute shigella and the traditional PCR assay was 620 CFU/mL. The LAMP detection limit was 42 CFU/mL in raw meat. The improved FTA-LAMP assay enables shigella to be detected in less than 2h in raw meat (contain DNA purified,LAMP and UV transilluminator).Conclusion: LAMP amplification using FTA filters provides a faster and more sensitive method for shigella detection than the standard cultivation and PCR method and it has higher specificity. For the rapid detection of shigella in food build a technology platform.