Screening Of The Aptamers Against Listeria Monocytogenes And Its Preliminary Application

Listeria monocytogenes is an intracellular pathogen.It widely exists in nature environment and has strong tolerance to adverse growing conditions. It even can grow at4℃and easily contaminate foods stored at low temperature. Because of these characters, Listeria monocytogenes can cause mortality rate as high as20%and is paid to widespread attention. Most governments develop safety limitation and standard assay to monitor Listeria monocytogenes in food. The detection method of Listeria monocytogenes can be divided into4categories:traditional culture and isolation, molecular biology technology, instrumental analysis. The detection of microbial population status with immune assay is important, which bases on the reaction of antigen and antibody,so antibody is the core reagents in immunology method. But people found some problems in the process of immunological methods.These defects display long time wasted to get biology antibody, inhomogeneous quality between different batches, and unstable property. Some researchers found a new ligand named aptamer which had similar antibody’s characters. The aptamer is easily modified, and can be synthesized unlimited. Many researches have showed aptamer would be hope of replacing biology antibody in the field of diagnosis and pathogen detection. In this research the single strain DNA (ss DNA) aptamers against Listeria monocytogenes were selected by systematic evolution of ligands by exponential enrichment (SELEX), and then the aptamers were preliminarily used to developed enzyme-linked aptamer sorbent assay(ELASA) to detect Listeria monocytogenes.The aptamers were screened by SELEX after15rounds. The primer labeled with biotin was used to clone sub-library which was harvested by magnetic beads linked streptomycin. At the same time the sub-library was amplified by primer labeled with digoxin in order to analysis the affinity displayed with OD405nm between aptamers and Listeria monocytogens. The results showed that the magnetic microspheres could effectively bind DNA and improve the efficiency of screening. The OD405nm gradually increased from0.051to0.69after15rounds of screening. The twenty-three aptamers obtained had no obvious homology according their sequences by sequence alignments. The aptamers were divided into4families according to the secondary structure predicted by RNA structure software. Four aptamers belonging to the fourth family had good affinity. No.21aptamer and No.35aptamer were selected to react with other bacterium. The results showed that the No.21aptamer and No.35aptamer had good specificity.Aptamers with good affinity were selected to establish sandwich ELASA for the detection of Listeria monocytogenes. The aptamers labeled with avidin as the capture ligand were adsorbed to micro-plate by combined with streptavidin. The sample and another aptamer with avidin were added in turn. Streptavidin with horseradish peroxidase was binded with the second aptamer. OD450nm value was determined after TMB substrate added. The results showed per well of micro-plates should be coated with6μg/mL streptavidin by drying method and40nmol capture aptamer. The optimal coloring time of enzymatic reactions was15min. The bacteria had serial10-fold dilution to detect the sensitivity of the method.The results showed that the limitation of this method was104CFU/mL. Listeria and other bacteria were detected by ELASA, when P/N value was greater than or equal2.1the sample was judged as positive, otherwise negative. ELASA could specifically detect Listeria monocytogenes. After the detection of milk samples which were contaminated with Listeria monocytogenes, Listeria monocytogenes can be detected after the enrichment of8h.In this study,23aptamers were screened by SELEX, two of which were used in sandwich ELASA.The results indicated this method could specifically detect Listeria monocytogenes and had good sensitivity. It indicated the aptamer had great potential as new ligands to apply in bacteria detection. This was a reference for aptamer screened against other bacteria and detection of pathogenic microorganisms.