The Detection Of 16SrRNA Gene Of Foodborne Pathogens By Real-time PCR Method

For the past few years, food safety has become a common topic of universal concern because of food safety incidents occur frequently. Food safety issues are related to the various levels of people's livelihood, food poisoning caused by the food-borne pathogens are the main aspects of food safety of various types of food safety issues.The most common food-borne pathogens involve Shigella, Salmonella and Staphylococcus aureus. The detection methods for food-borne pathogens mainly follow the traditional identification method of bacteria isolates and cultures, but the shortage is complicated, low sensitivity and only confirms or rules out one pathogen, so that it is difficult to meet the needs of fast and accurate detection of food-borne pathogens. Therefore, it is very necessary to establish a multiple, fast and effective detection techniques to meet the needs.In this study, a multiplex real-time PCR detection technology for the three food-borne pathogens Shigella, Salmonella and Staphylococcus aureus had been established utilizing the TaqMan probe. Using 16 S rRNA gene as a target gene to design primers and TaqMan probes for real-time PCR, the dual control the select of the target gene, with good specificity and sensitivity.The main results were as follows:(1)PCR primers were designed using16 S rRNA gene as a target gene, the three bacteria Shigella, Salmonella and Staphylococcus aureus as detection objects and six kinds of bacteria as control bacteria have been detected. Sequencing of the fragment from conventional PCR amplifying had confirmed that the primers can be used as degenerate primers for the further study.(2)Establishing and Optimizing a single real-time PCR detection system aimed to detect single bacterial gene, and on the basis of system, further optimizing and establishing a triple real-time PCR detection system.(3)the Specification of real-time PCR system is fine, only the target strains appear positive amplification signal.(4)High sensitivity, the sensitivity of the genomic DNA was 10-4 ng.(5)Standard plasmid was constructed successfully, and standard curve for the three bacteria have been finished.The multiplex real-time PCR method for Shigella, Salmonella and Staphylococcus aureus have been established, it is simple and efficient, greatly reducing the detection time. It has a good prospect in the rapid screening for foodborne pathogens.