Visual Rapid Detection Of Common Pathogenic Bacteria In Infant Formula Milk Powder And Assembly Of Kit

Infant formula milk powder is a kind of infant food made by adding nutrients similar to breast milk according to the special constitution of infants.It is the main source of nutrition and the composition of dietary structure of infants.It is easy to be polluted by all kinds of microorganisms because it is rich in nutrients.In recent years,there have been frequent safety problems in the field of infant formula milk powder.The occurrence of safety events mainly focused on infant foodborne diseases caused by the pollution of pathogenic microorganisms,which makes all sectors of society pay more and more attention to the safety of this special food.Among them,Salmonella,Enterobacter sakazakii and Staphylococcus aureus are the microorganisms that must be tested in the national food safety standard infant formula food GB10765-2010.The multiple loop mediated isothermal amplification kit assembled by the research institute can carry out visual and rapid detection for the above three pathogens,and the detection can be completed in about 2 h.Without complex temperature changing equipment,the detection results can be judged intuitively by the naked eye,which greatly shortens the detection time,reduces the restrictions of equipment and site,and can contribute to ensuring the food safety of infants and young children.In this study,three pairs of specific primers were designed by using the inv A gene of Salmonella,omp A gene of Enterobacter sakazakii and nuc gene of Staphylococcus aureus.Optimizing various reaction conditions,a visual rapid detection method of Salmonella,Enterobacter sakazakii and Staphylococcus aureus in infant formula with simple operation,high sensitivity and strong specificity was established by using ring mediated isothermal amplification（LAMP）.Firstly,various reaction conditions were optimized,and hydroxynaphthol blue（HNB）indicator was added into the reaction system.The results of LAMP amplification can be observed by naked eyes.A single LAMP visual detection method for Salmonella,Enterobacter sakazakii and Staphylococcus aureus in infant formula milk powder was established.On this basis,a multiple LAMP visual detection method for the simultaneous detection of Salmonella,Enterobacter sakazakii and Staphylococcus aureus in infant formula milk powder was optimized and established,and a multiple LAMP detection kit was assembled to realize the visual and rapid detection of Salmonella,Enterobacter sakazakii and Staphylococcus aureus in infant formula milk powder.Finally,through the determination of the sensitivity of the kit,it was determined that the detection limits of the multiple LAMP kit for the genomes of Salmonella,Enterobacter sakazakii and Staphylococcus aureus were1.03×10^-8ng/μL,1.24×10^-7ng/μL、9.17×10^-7ng/μL.The detection limits of multiple LAMP kit for Salmonella,Enterobacter sakazakii and Staphylococcus aureus were 2.6×10⁰CFU/m L,4.1×10¹CFU/m L,5.5×10⁰CFU/m L.The detection limits of Salmonella,Enterobacter sakazakii and Staphylococcus aureus in artificially contaminated samples were 1.09×10^-9ng/μL,1.13×10^-8ng/μL,9.97×10^-7ng/μL.The assembled multiple LAMP kit is applied to the detection of actual samples.The results are compared with PCR method and national standard method.It shows that the specificity,the sensitivity of the kit meets the relevant standards,and the detection time can be completed in about 2h.The detection results can be directly judged by the naked eyes without complex equipment and instruments,which meets the requirements of on-site rapid detection.