Rapid Detection Of Bacillus Cereus Using Loop-Mediated Isothermal Amplification

Bacillus cereus is a widesPread baeterial pathogen that can infect animinals and human beings, food much easy be polluted after exposed more than 2h.The foods most prone to contamination by Bacillus cereus are milk,rice and food to contain protein.In recent years,there have been increasing reports of Bacillus cereus related food poisoning.Increasing rates of detection methods that are faster,more specific and high sensitive.Current standard detection mehtods require lenghty culture enriehment steps to increase target bacterial numbers before isolation and identification can be performed.Loop-mediated isothermal amplification(LAMP)is a novel nucleic acid amplification method developed by Eiken Chemical Co.,Ltd.,Japan,and has the potential to replace PCR because of its simplicity,rapidity,specificity,and cost-effectiveness.The LAMP assay has emerged as a powerful gene amplification tool for the rapid identification of microbial infections and is being increasingly used by various investigators for rapid detection and typing.Bacillus cereus is known to cause two different types of food poisoning,which are characterized by either emesis or diarrhea.At present,two different protein complexes,each consisting of three exoproteins,as well as a single protein(cytotoxin K),are discussed as causative agents.The specific LAMP primers of Bacillus cereus were designed on the basis of the published sequence of strain hblA(GenBank accession number AJ237785)with the LAMP primer design support software program,using primer explorer software.The best concentration of primer,the concentration of BST,the concentration of Mg²⁺,the concentration of betaine。The LAMP reaction was carried out in a 25μl total reaction mixture volume with containing 40 pmol each of inner primers FIP and BIP,5 pmol each of outer primers F3 and B3,1.0 mmol/L each deoxynucleoside triphosphate,1.0 mmol/L betaine,6.0mmol/L MgSO₄,2.5μL Bst DNA Polymerase Buffer(20 mM Tris-HCl(pH8.8,25℃),10 mM KCl,10 mM(NH₄)₂SO₄,2 mM MgSO₄, 0.1%.Triton X-100),8 U of Bst DNA polymerase,and the specified amounts of target DNA.The mixture was incubated at 62℃for 60 min in a heating block and then heated at 80℃for 2 min to terminate the reaction.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.An assay using Loop-mediated isothermal amplification technology(LAMP)was developed for directly,rapidly detection of Bacillus cereus.The specific LAMP primers of Bacillus cereus were designed on the basis of the published sequence of strain hblA and the gene was purified by FTA filter, also FTA-LAMP was used both in Bacillus cereus and in pasteurized milk of Bacillus cereus.Under the optimum reaction conditions,a rapid technique for detection of Bacillus cereus was initially established by LAMP.The LAMP assay was found to be 10-fold more sensitive than the traditional PCR assay, detecting 4.4cfu/mL of Bacillus cereus.The LAMP detection limit was 5.7cfu/mL of pasteurized milk. The improved FTA-LAMP assay enables Bacillus cereus to be detected in less than 2 h(contain DNA purified,LAMP and UV transilluminator).Conclusion:LAMP amplification using FTA filters provides a faster and more sensitive method for Bacillus cereus detection than the standard cultivation and PCR method and it has higher specificity.For the rapid detection of Bacillus cereus in food build a technology platform.In this study,LAMP was made.The result indicates:the sensitivity of the LAMP assay is 100%, the specificity is 72.7%,and the coincidence rate is 94.5%.