Real-time Fluorescence Loop Mediated Isothermal Amplification For The Detection Of Listeria Monocytogenes In The Food

Listeria monocytogenes is a major pathogen Listeria, hardy,, can grow beyond a widetemperature range and widely in nature, is one of the most deadly foodborne pathogens.Eating the food contaminated with L. monocytogenes can cause humans and animalsmeningitis, sepsis, pregnant woman would lead abortion, the mortality is as high as30%~70%. L.monocytogenes had caused a lot of the outbreak of food safety incident around theworld each year.and in the recent years, China also appeared the Introduced meat fromabroad was bacterial infections many times. Due to the presence of a little Listeria in foodand the interference of the other micro-organisms, it appears some difficulties in thedetection of Listeria.Therefore, the establishment of a reliable method for detectingL.monocytogenes rapidly in food is necessary. Traditional detection methods are outdated,and can not adapt to meet the modern requirements of rapid detection. Some betteridentification and control techniques have been a great concern.Loop-mediated isothermalamplification (LAMP) is a kind of novel nucleic acid amplification techniques in vitro,with the characteristics of simple, fast, strong specificity and high sensitivity, which hasbeen applied to the detection of pathogens, genetically modified food testing, medicalapplications and many other areas.The real-time fluorescence Loop-mediated Isothermal Amplification(RTF-LAMP)combine LAMP with fluorescence using a new instrument named ESE-Quant TubeScanner connected to the computer to conduct a test,which monitor real-time the LAMPdigital amplification conditions. We called this method as the real-time fluorescenceLoop-mediated Isothermal Amplification techniques, RTF-LAMP for short.The study adopts inlB genes for test target using the online primer design software todesign primers, and using scanner reaction60min at62℃to test the RTF-LAMP digitalamplification conditions. Finalize RTF-LAMP reaction system is: FIP and BIP primerseach3.5μmol/L in the reaction, the primers F3and B30.5μmol/L in the reaction, theother reaction components were0.3mM dNTPs,1mM MgCl2,2.5μL10×Bst enzymebuffer,1μL800U BstDNA polymerase,1μL template DNA, betaine0.5mmol/L,1:400SYBR Green I0.5μL, sterilized double-distilled water make up the volume to25μL.The specificity of this method was verified with3L.monocytogenes strains and theother17non-L.monocytogenes strains. The results indicated L.monocytogenes was positive and other strains were negative.Due to L.monocytogenes is the main pathogens leadingthe disease in the refrigerator, where people was used to place cooked food. RTF-LAMPtechnology rapidly detecting L.monocytogenes was explored and the sensitivity, artificialcontamination detection limit were determined in this study.So this RTF-LAMP design toartificially pollute bacon with L.monocytogenes, and the detection limit is35CFU/mL, andthe detection sensitivity is11CFU/mL. Compared with LAMP, its sensitivity to10timeshigher, and compared with PCR method,104times higher than that of its sensitivity.This research established an RTF-LAMP method with sample of L.monocytogenesdirect detection. Compared with traditional training methods, RTF-LAMP cost1hour tocomplete the L.monocytogenes detection. RTF-LAMP has the advantage of saving time,simple operation, but also can greatly prevent contamination. For the rapid detection ofL.monocytogenes in food RTF-LAMP build a technology platform.