The Usage Of PCR Method To Detect LM And EHEC O157:H7 In Food

Food safety is a major public health problem, which directlyconnects with people's physical health and social stability. ListeriaMonocytogenes(LM) and Enterohemorragic E.coli (EHEC)O157 ∶ H7are food borne pathogens who have huge impact on the Public Health.Food and health administrations of each country have already paid greatattention on the harm induced by the two pathogens recently. Trying tofind a rapid, sensitive, specific and reasonable testing method becomesan important problem to be solved by Food and health administration inthe world. Nowadays, the methods used in Primary Department of ourcountry are still complicated and time-costing (a testing report needsabout 3-14days usually) which applied the traditional isolation, cultureand biochemical identification way. As the development of molecularbiology, polymerase chain reaction (PCR) testing way is applied in thetesting of food borne pathogens, which is used in this study. Aiming atthe pathogenic characters of LM and EHECO157 ∶ H7, with the specificprimer selected, a rapid PCR method of testing the LM andEHECO157∶ H7 in food is built, in order to improve the sensitivity,decrease the testing time to avoid the food poisoning caused by the twopathogens and support technique to diagnosis food borne disease.This investigation was divided into two parts. The first part was tobuild the PCR testing method of LM. A pair of primers was designedand complemented to the hemolysin A gene (hlyAgene), which is ownedby LM only and has highly conservation. The target fragment amplifiedis 706bp. While the PCR method was built, the reaction conditions wasoptimize and then the sensitive test and specific test was carry out .Itshows that the method has high sensitivity and the limitation of thismethod is 3.3pg/μL DNA of LM.Samonella, Bacillus.Cereus,Staphylococcus .aureus and E.coli were selected to demonstrate thespecificity of the PCR which can be finished in 24 hours. And the resultsshow that the positive control can amplify the target fragmentspecifically, while the others did not. That proves this method hasmore specificity. 480 food essays were tested by this method. Theresults are as follows: 79 samples of LM are positive (positive rate16.46%), 240 cases of Meats account for 23.75%( 57/240),of whichchickens with the highest positive rates account for 33.00% ,while thelower rates of vegetables and fishery products are 2.5% respectively.So does the using PCR methods.Then the 79samples of LM positiveessays are cultured. The coincidence rate is 100%between the results ofthe PCR and the microbiologic culturation. In a word, the first part ofthe method is more sensitive, specific, rapid and brief than traditionalways. It is better to applied to rapid food test.The second part is supposed to build multiple testing method ofEHEC O157 ∶ H7. three pairs of primers designed and complemented tothe Shigella toxin gene (stx), O antigen gene (rfbE), H antigen gene(fliC) respectively. After the multiple amplification under PCR wasbeing processed, corresponding target fragments are acquired, whoselength are 228bp,378bp and 709bp respectively. It is proved that themost optimal primer dosage in 30ul reaction system is rfbE 0.3μL,fliC0.4μL,stx 0.3μL both the upetream and the downstream primers, themost optimal enzyme dosage is 0.3μL, the most optimal dosage of dNTPis 5.0μL and the most optimal annealing temperature is 50.7℃ afterrefine the multiple PCR reaction conditions e.g the dosage of primers,TaqE, dNTP and the parameters. 480 cases of various food essays weredetected with multiplex PCR methods and 1 cases showed the EHECO157 positive ,the rate is 0.21%. Belong to EHEC O157 sero-groupwhose H antigen is not known. Multiple PCR testing method is morerapid and economic than that of traditional ways and simple PCR. It isof greater significance in the supervising and controlling of the foodsafety.