Rapid Detection Of Listeria Monocvtogenes By Real Time PCR Technology Combined With Immunomagnetic Beads

Food safety is the major public health problem caused by toxic and harmful substances in food. Foodborne pathogens are the most important source of food safety issues. Listeria monocytogenes is one common bacterium among the foodborne pathogens, which causes a series of foodborne listeriosis as septicemia, encephalitis, meningitis, abscesses and miscarriage, and the mortality rate is high as30%to70%. In recent years, the traditional cultural method has disadvantages of time-consuming and labor-intensive, while conventional PCR method has long pre-processing time, easy to polluting the environment and other shortcomings of the experiment. Therefore, developing a rapid, accurate and reliable method for detecting Listeria monocytogenes in food is very important and meaningful.In this study, immunomagnetic beads separation technology was applied for rapid enrichment of bacteria from contaminated samples containing bacteria, instead of traditional culture enrichment methods. Combined with real-time PCR detection technology, it improves the detection rate of Listeria monocytogenes, shortens the test time and improves the detection efficiency, providing a more rapid and convenient method for the market quarantine inspection.In preparation process of immunomagnetic beads, comparing the effect caused by concentrations of EDC/NHS, activation time of EDC/NHS, concentrations of antibody, coupling time, and coupling temperature, finalized the specific factors for the preparation of immunomagnetic beads. The immunomagnetic beads were washed with PBST (pH=6.0), adding475u L PBST (pH=7.4) and25μL1mg/mL antibody, incubated at room temperature for2h, then separated, and blocked. In addition, comparing the amount of immunomagnetic beads, enrichment time, and pH value of scrubbing liquid in the detection process, the optimum conditions were determined:the immunomagnetic beads containing100μL were added into100μL of the bacterial culture medium, and incubated for1h at28℃(180rpm), then placed in the magnetic holder for separation, washed with500u L of PBS (pH=7.4) for three times, and finally with50μL PBS (pH=7.4) resuspended. The optimized factors for real-time PCR were as follows:Premix Taq:12.5 μL, ROX reference Dye:0.5μL, forward primer:1μL, reversed primer:1μL, Taqman probe:1μL, double distilled water:4μL, DNA template:5μL, and the total volume of25μL. The temperature setting was:the first step95℃,2min; the second step95℃,5s,60℃,35s, for40cycles.The method showed good linearity at the concentration range of10-10CFU/mL, and the detection limit was80CFU/mL. The method was applied to four categories of nine kinds of foods:seafood(sea fish, shrimp), meat(raw pork, sausage), vegetables(lettuce, cucumbers, tomatoes), and diary(milk, cheese). The results showed that the immunomagnetic beads combined with real-time PCR detection method has advantages including high sensitivity, high accuracy and good reproducibility. Compared with traditional culture methods, our study reduce the detection time effectively, and improve the detection efficiency.