Development Of Detection For Staphylococcus Aureus In Milk Based On Immunomagnetic Beads And Loop-mediated Isothermal Amplification Method

Quality and safety of dairy products is the major concern of food safety issues, it relates to human health and economic development. Staphylococcus aureus (S. aureus) is a bacteria which exists everywhere in nature can cause zoonosis, food poisoning caused by S. aureus has become a worldwide health problem, especially in recent years, the dairy issues are getting more and more attention. However, the conventional detection method is time-consuming, low sensitivity, complicated operation and can not meet the demand for rapid detection of primary workstation, and milk composition is very complex, fat, protein, metal ions will interfere the test results, therefore, eliminate the interference factors in milk and establish a rapid, sensitive, reliable and specific detection method of S. aureus in milk have great significance.First, the polyclonal antibody of S. aureus were preparated, identificated and purified. Then immunomagnetic beads were made for separating S. aureus in the milk. Salmonella typhimurium,Shigella dysenteriae,Bacillus cereus,Bacillus proteus,Escherichia coli and E. coli O157: H7 were used as negative control to verify the specificity of immunomagnetic separation. The milk was infected with different concentrations of S. aureus, through this method to determine the sensitivity is 2.81×102CFU/mL.Using LAMP method, the LAMP primers were designed by Primer Explorer V3.0, to detect the heat-stable nuclease gene (nuc) of S. aureus. Several important parameters were chosen for the exploration and optimization of LAMP, ultimately, the reaction system was determined: 2.5μL10×buffer, 0.6mMdNTP, 0.2μM outer primers, 1.6μM primers, 0.8MBetaine, 4mM MgCl2, 1μL Bst polymerase, 1μL template. The specificity of LAMP method was validated, and the sensitivity is 9.4×101CFU/mL.Finally, immunomagnetic separation and the LAMP method, which is IMS-LAMP method, were combined to detect S. aureus in milk. At the same time, milk samples were collected for practical application. 200 milk samples were tested, compared with the traditional national standard method, the results had no significant difference, stable and reliable, the detection limit was 5.0CFU/mL.In this study, immunomagnetic separation and loop-mediated isothermal amplification technology were combined to detect S. aureus in milk, a specific and sensitive method for the rapid detection of S. aureus was developed, which is a theoretical reference of rapid assay kit.