Rapid Detection Of Listeria Monocytogenes By Using Specific Immunomagnetic Beads

Aims: To combine immunomagnetic separation technique with selective medium methodby producing Listeria monocytogenes specific immunomagnetic beads, with the purpose of theseparating and identifying Listeria monocytogenes. Meanwhile, immunomagnetic separation isapplied to conduct sample pre-treatment, which combines with loop-mediated isothermalamplification to establish a rapid detection method on Listeria monocytogenes.Methods: Asctic type monoclonal antibody was produced and purified using hybridomas(1B10A7) against Listeria monocytogenes. Three types of magnetic beads with different surfacefunctional groups were selected to prepare immunomagnetic beads. By comparing their captureefficiency, the optimal one was chosen to help establish the immunomagnetic beads-selectivemedium method. Analysises including the specificity, sensitivity and milk sample tests wereconducted to assess the detection effects. Later on，based on the hly and prfA virulence genes ofListeria monocytogenes, four pairs of primers which would be applied in loop-mediatedisothermal amplification were designed. Undertaking positive screening, cross reaction screeningand fluorogenic quantitative PCR, the optimal pair were picked out as well as identified bymolecular cloning after PCR. Immunomagnetic beads-loop-mediated isothermal amplificationwas established and assessed through sensitivity analysis and milk sample tests.Results: The titer of purified monoclonal antibody against Listeria monocytogenes was6×10~4, when coupling with immunomagnetic beads, the tosylactivated magnetic beadspurchased from DynabeadTMwere selected to process the following trials. Method combiningspecific immunomagnetic beads with selective medium managed to detect Listeriamonocytogenes of a concentration of103CFU/mL or above despite a little cross reaction withListeria innocua. After six hours of bacteria enrichment, milk samples were successfullydetected, which was42hours less than regular enrichment, with a limit of detection of0.7CFU/mL. An ideal pair of prfA specific primers was selected because of its high specificity andefficient amplification, its amplified product was accorded with the target sequence. Thedetection sensitivity of immunomagnetic beads-loop-mediated isothermal amplification was10~1CFU/mL and its limit of detection on milk sample was1CFU/mL. Conclusions: The immunomagnetic beads-selective medium method was able to finishdetecting milk sample within30hours, which was42hours less than national standard methodwith the same sensitivity. The immunomagnetic beads-loop-mediated isothermal amplificationpossessed strengths including high specificity and sensitivity, as well as rapid detection, it coulddetect sample of low concentration of positive bacteria without pre-enrichment step. This studyhas established rapid detection methods on Listeria monocytogenes by combining withimmunomagnetic separation, it is hopeful that they will be applied in food safety monitoring inthe future after carrying out the condition optimizations.