Development Of A Rapid Method For Detecting The Genes Of Three Pathogenic Bacteria In Milk

Dairy products contain many nutrition components,which are important food varieties in people’s Daily life.The quality and safety of dairy products are directly related to the health of consumers and the healthy development of the dairy industry.The total number of bacteria and pathogenic bacteria in dairy products directly effect the quality of dairy products.The national standard requires that the total number of bacteria in raw milk should not exceed 2×10⁶ CFU/mL,while the total number of bacteria in pasteurized milk should not exceed 1×10⁵ CFU/mL Salmonella and Staphylococcus aureus are the most frequently isolated pathogens causing food poisoning.The requirements of the national standard of these two pathogens in dairy products are not detectable.However,the national standard has a long time-consuming to detect the total number of bacteria and the specific pathogens,which is not conducive to the screening and on-site detection of enterprises.Therefore,a rapid and efficient method is urgently needed.The microorganisms in dairy products mainly contain bacteria and yeasts.According to the previous research in our laboratory,the bacterial components in dairy products in Jilin Province were analyzed and determined.Real-time quantitative PCR（qPCR）was used to obtain the corresponding relationship between the CT value of different bacteria and the total number of bacteria.We found that the standard curve value of E.coli 16S rDNA could be used to measure the total number of common bacteria in dairy products.Based on this result,a qPCR method was established to detect the total number of bacteria in dairy products.The total number of bacteria in dairy products was determined by establishing standard curve and determining the CT value.The actual samples were tested by comparing with the national standard method,so as to determine the accuracy of qPCR and identify the quality of dairy products more quickly and efficiently.Firstly,this study constructed the recombinant plasmid,established the standard curve and determined the CT value standard,which can quickly detect the total number of bacteria in dairy products,testing complete within 3 hours,the detection result of the actual milk samples is good,the accuracy rate is about 95.83%,which solved the problem that the national standard method can’t detect the total number of bacteria quickly and efficiently.In addition,the detection of pathogenic bacteria in food is also the focus of this study.In this study,loop-mediated isothermal amplification（LAMP）was established to qualitatively detect Salmonella and Staphylococcus aureus in dairy products.Firstly,the LAMP methods were established to individually detect Salmonella and Staphylococcus aureus by optimizing the reaction conditions.Secondly,the rapid and simultaneous visualization detection methods was established for simultaneous detecting Staphylococcus aureus and Salmonella.Finally,the LAMP kits were assembled according to the national standards.In terms of detection of pathogenic bacteria in dairy products,the primers were designed and screened,then In this study,by designing and screening specific primers,and then optimizing different conditions,a single LAMP detection method was established to detect Salmonella and Staphylococcus aureus,the results showed that the detection limit of Salmonella was1.55×10^-88 ng/μL and Staphylococcus aureus was 7.8×10^-6 ng/μL by addition standard samples.Then,simultaneous LAMP detection method was established to optimize the conditions and assemble kits to detect the actual samples.Compared with the national standard method and PCR method,the sensitivity and specificity of the kit accrods with GB standards,the detection can be completed within 2 hours and visualized.This method uses simple equipment and instruments,which is suitable for rapid detection of enterprises and large-scale samples on-site.