Establishment Of Two Rapid Detection Methods Of Listeria Monocytogenes And Development Of The Kits

Listeria monocytogenes is a gram-positive bacillus,which is an important food-borne zoonotic pathogenic microorganism,which can cause listeriosis in humans and various animals.The World Health Organization has listed it as the 20 th century,one of the four food-borne pathogenic microorganisms.In recent years,the occurrence of Listeria monocytogenes worldwide is on the rise,and the pollution in food is widespread.At present,the traditional method of detecting Listeria monocytogenes in food is complicated and the detection period is long,which can not meet the need of rapid detection of Listeria monocytogenes in food.Therefore,it is necessary to establish a rapid and convenient detection method for Listeria monocytogenes.In this study,four specific loop-mediated isothermal amplification(LAMP)primers were designed for Listeria monocytogenes(L.monocytogenes)hly A gene,And the reaction conditions were optimized.The rapid detection method of Listeria monocytogenes was established.The results showed that the method could specifically detect Listeria monocytogenes with the detection limit of 3 CFU / m L.The amplification products can be determined by agarose gel electrophoresis and color reaction.LAMP can detect Listeria monocytogenes quickly,intuitively and accurately,and it is a kind of suitable detection method for grass-roots field application.The Listeria monocytogenes could be detected at 63 ? for 45 min,the detection limit was 3 CFU / m L,the specificity was 100% Positive and false negative appear.The stability and reproducibility of the kit were verified by 20 batches of samples.The results showed that the whole process of LAMP detection(including sample pretreatment)2h.It is suitable for rapid detection of Listeria monocytogenes.In this study,we also developed a rapid and convenient method based on loop-mediated isothermal amplification(LAMP)and Lateral flow dipstick(LFD)Of Listeria monocytogenes detection method.Four specific primers and one Fluorescein isothiocyanate(FITC)-labeled probe were designed for the Listeria monocytogeneshly A gene.The biotin-labeled LAMP amplified product can specifically hybridize to the FITC-labeled probe,and the hybridization product is detected by LFD.The optimal temperature and time for the reaction were 63 ? for 50 min,and the sample was pretreated for 80 min.LAMP-LFD method can be specifically detected Listeria monocytogenes,the other six common food-borne pathogens were detected negative.The detection sensitivity for pure bacterial cultures was 3.6 CFU / m L,which was100-fold higher than that of the PCR method using external primers.The results show that the LAMP-LFD method can quickly and specifically detect Listeria monocytogenes.It is simple and economic,and does not need other auxiliary instruments.The results can be directly judged;suitable for primary laboratory,emergency detection or on-site monitoring.Use,with a high promotional value.