Study On The Rapid Detection Of Four Food-borne Pathogens In Food By Multiplex PCR

The food security is a momentous public safety problem of the whole world. It not only affects people's health, but also relates to the national economy and the people's livelihood. In these problems, the food poisoning result from bacilli was the most harmful. According to data came from hospitals and inspection-quarantine departments, the main bacterial pathogens were Salmonella, Staphylococcus aureus, Listeria monocytogenes, and Shigella and so on. The symptoms usually were fever, headache, nausea, vomit, bellyache and diarrhea, and even appeared spasm, desudation, collapse and hypopiesis, so much as shock or die. On the other hand, the traditional detection methods were based on cultivation of microbes, selective separation and biochemical identification which took much time and work with low sensitivity and sham negative result. Immunological technique was advanced but also wasted time and had large errors. As to the technique using DNA probe, it'was very cost and inconvenient. In order to prevent and control the food security accident, it's very necessary to develop a food-borne pathogenic microorganism detection method that is rapid, exact and economical.Methods:(1) Amelioration of the extraction of genome DNA and the pretreatment of the food sample to reduce cost, time and processes;(2) Aiming at the four types of food bacterial pathogens (Salmonella, Staphylococcus aureus, Listeria monocytogenes, and Shigellosis), we selected the Salmonella invaded protein gene (invA), Listeria monocytogenes hemolysin gene (Hly), Staphylococcus aureus heat-resistant ribose gene (nuc) and Shigella invaded plasmid antigen gene (ipaH) for our targets, and designed four pairs of specific primers, then optimized the PCR system and conditions to establish the multiplex PCR rapid detection method of these four types of microbes;(3) Carry out the artificial infection of food samples, detect the food on sale and validate with the traditional methods to checkout the reliability of our method. Results:(1) The advanced method for extraction the genome DNA with guanidine thiocyanate was rapid, economical and effective;(2) FAT filtration and gradient centrifugation could abate the affection of sample's ingredient in PCR before the detection;(3) The four pairs of primers appeared high specificity and did not interfere with each other;(4) The multiplex PCR system to detect these four types of microbes was steady;(5) The time of whole detection procedure was less than 10 hours, and the detection limit was few as 1 CFU/mL after 4 to 8 hours bacterial culturation..Conclusions:The multiplex PCR method was simple, rapid, exact, sensitive and applicative; it could be used in the fields such as food sanitation detection and clinical inspection.