Application Of Real-time Fluorogenic Quantitative PCR Assay To Rapid Detection Of Listeria Monocytogenes In Fresh Agricultural Products

Listeria monocytogenes,which is a notorious foodbome pathogenic bacteria,has a fatality rate is as high as 30%.In recent years,reports of Listeria monocytogenes infected in fresh agricultural products often appear.Especially fresh cut finit and vegetable and Salmon as ready-to-eat(RTE)products,vulnerable to microbial pollution problems.So as it,this study,selected fresh cut melon and Salmon as experience materials,establishes the rapid detection of Listeria monocytogenes through the RT-PCR with SYBR Green ? and TaqMan Probe.It aims to establish a simple,fast and selective,high sensitivity detection method.The method enhances the safety of fresh agricultural products.The research content mainly includes the following several aspects:(1)According to hly gene of Listeria monocytogenes,the author design its mergers primer and probe,and its feasibility was proving.Meanwhile,the author establishes the RT-PCR method detecting Listeria monocytogenes.The results show that sequence homology was 100%,and the merger primers which were designed by fluorescent quantitative PCR can amplify the specific electrophoresis banding.And the feasibility was good.(2)Comparing the advantages and the disadvantage of two methods,called "bacterial genomic DNA extoction kit" and "magnetic beads bacterial DNA extraction kit",the first one is better.(3)For determining the sensitivity of the RT-PCR,the author builds the hly-plasmid standard,and establishes the standard curve.The standard curve of SYBR Green ? was establish,and R2=0.9993,linear equation was y=-3.73+44.696 x,and R2=0.9955,linear equation was y=-3.439+40.36 x.R2 for TaqMan probe was 0.9912,linear equation was y=-4.313+52.182 x.The sensitivity of plasmid for SYBR Green ? was 7.69 × 10 copies/?L,1.12 copies/?L,and the sensitivity of plasmid for TaqMan probe was 1.12×102 copies/?L.(4)Accomplishing the detection of Listeria monocytogenes in the artificial polluted melon and salmon by the method above,the author makes sure the minimum detection limit of this method and established the standard curve.The results show that 6.28×10 cfu/mL is the detection limit of SYBR Green ? in the artificial polluted melon.Established the standard curve,its formula is y=-3.19x+37.38,and its correlation coefficient is 0.9958.And 3.5×102 cfu/mL is the detection limit of SYBR Green? in the artificial polluted salmon.Established the standard curve,its formula is y=-3.087x+39.503,and its correlation coefficient is 0.9955.6.28×102 cfu/mL is the detection limit of TaqMan probe in the artificial polluted melon.Established the standard curve,its formula is y=-4.331x+49.693,and its correlation coefficient is 0.9939.