Rapid Detection Of Enteropathogen With PCR Assay

The problem of food safety is becoming more awaked and more serious for people in many counties. It is associated closely with the national development and people's life job and health. The mostly factor of food poisoning is bacterial food poisoning. The one of reasons causing human being disease is enteropathogen in food. The traditional detection of pathogeny microorganism need a long time,operation with trivial details.Meanwhile,the traditional detection can't get a perfectly identity result from some bacteria. There are more and more problem in our practice. So, we study the best advanced and sensitive PCR technology presently to develop a rapid,simple,precise quantity,sensitivity,speciality,economic detecting method. We study the familiar Enterobacteriaceae in our food to get a efficacious approach of food poisoning diagnose and medical treatment detection. In paper,there are Enterobacteriaceae including Enterotoxigenic E.coli, Enteroinvasive E.coli, Shigella spp,Salmonella spp.There are two or more pairs of primer in multiplex PCR.It can amplify several DNA segments in a PCR. Multiplex PCR is effective,systemic and economic detecting method.The study applied multiplex PCR for detecting or identity a great deal of pathogeny microorganism. The special strain or genus gene sequence are screened out. According to principle of primer designed,we progress to design primer. Then we amplify gene sequence of kinds of bacteria in a PCR. The multiplex PCR system is optimized by adjusting the concentration of MgCl₂ and primer,the quantity of Taq enzyme and template,the anneal temperature and cycle parameter. These primer of gene sequence can amplify their DNA segment respectively. This multiplex PCR system is special and sensitive as well as the single PCR. The random combination is successful. We spend the same time amplifying in the multiplex PCR and in single PCR. The entire course from sample managed to result need 2-3 hours. The result of detecting sample accord with the detection standard method of food sanitation microorganism. The stability and repetition of assembled reagent is perfect. So,the reagent is applied value.It can be applied in practice.The strongpoint of real-time PCR is high sensitivity, strong speciality, nothing of management after PCR reaction. Real-time PCR extensively apply in food and clinic detection presently. The primer and probe based on coding DNA segment of Enterotoxigenic E.coli heat-stable enterotoxin I gene and Enterotoxigenic E.coli heat-labile enterotoxin I gene are designed, using MGB-Taqman probe and ordinary Taqman probe respectively. We make the positive standard preparation,exploring reaction system and condition of real-time PCR. The reaction curve of real-time PCR is protracted by detecting fluorescence intensity.Because Ct value and logarithm of original template quantity exist in linear relation,we can carry through absolutely quantity of sample according to the standard curve. The real-time PCR is special and sensitive. The veracity of detecting sample accord with comparative method. The probability of leaving out detection isn't existence. The stability and repetition of assembled reagent is perfect. The entire course from sample managed to result need 2 hours. The method is more predominant than other method. With the further development of the method, it will become a optimal standard method of detecting Enterotoxigenic E.coli internationally.