Study Of Multiplex PCR Technology In The Simultaneously And Efficiently Detection Of Food-borne Pathogenic Bacteria

In recent years,with the rapidly progress of economy and technology,food and drugs safety problems have become more and more in the monitoring of food's and drug's pathogens in China.And dozens of pathogens had been detected.Due to traditional detection methods which take a long time and the operation is complicated.So we use multiplex PCR to improve the detection method to achieve fast and efficient foodborne pathogens.Multiplex PCR is the addition of two or more pairs of primers in the same reaction system and amplification of two or more templates.We selected strains provided by the Jilin Provincial Food and Drug Administration: Staphylococcus aureus,Salmonella paratyphoid B,Pseudomonas aeruginosa,Bacillus subtilis,Bacillus brevis,Escherichia coli,Aspergillus niger as the target strain.The first was to extract the genome of the pathogens,because Aspergillus niger was a fungus so the selected genome extraction kit was different from bacteria's.Then we designed their specific primers,and we also set the negative control group in the PCR amplification,observing by the agarose gel electrophoresis map.We could determine whether the two pairs of primers were cross-amplification.Finally,the multiplex PCR reaction was carried out using the milk.We screened and identified a total of 18 genes to seven pathogens,and the result of seven pathogens' s specific genes were Salmonella paratyphoid staphylococcus aureus,Escherichia coli's uid A gene,Staphylococcus aureus' s nuc gene,Bacillus pumilus' s gyr B gene,Bacillus subtilis' s rpo A gene,Pseudomonas aeruginosa's Opr L gene,Aspergillus niger's fum8 gene.The specific primers were designed with the specific primers designed for the purpose of these genes.The length of the primers was 665 bp,the primers of Paratyphoid Salmonella paratyphi was 500 bp.The primers of Bacillus subtilis were 403 bp,the length of Bacillus subtilis was 349 bp,the primers of Staphylococcus aureus were 280 bp,the length of Escherichia coli was 210 bp,and the primers of Pseudomonas aeruginosa were 105 bp.Then the optimized reaction conditions and reaction system were optimized,and the detection limit was 1.2 CFU/m L.Compared with the developed Ge XP-PCR technique,the detection limits of the six pathogens were 420,310,270,93,85,and 66 CFU/m L,increased by at least 50 to 350 times.And the multiplex PCR assay hads the advantages of low cost and good stability compared with other biosensor and immunological detection methods,while still could increase the number of strains.We used the multiplex PCR to test the initial application of milk samples,in which the detection limit of the other six pathogens in E.coli was 102 CFU/m L,and the detection limit of other 5 pathogens was 10 CFU/m L.However,we found that only E.coli can amplify the target band at a concentration of 104 CFU/m L.Possible causes: 1)In Chapter 2,it was mentioned that the multiplex PCR reaction should be performed on the reaction conditions and systems to fine adjustment,so the improvement of this experiment is to milk as a template should be fine-tuning condition and system,and then to test other types of samples,such as meat,soy products,as a template should be properly adjusted;2)It is also possible that the Tm values of the primer pairs of E.coli were 58.7 °C and 62.9 °C,which were higher than those of other primers,and this was the fact that the reaction conditions were not suited.So it was necessary to adjust the application of different foods reaction conditions.We also tested a kind of sterile eye drops provided by college of pharmacy of Jilin University and treated with sterile water as the negative control group.As a result,the eye drops were sterile and it took four hours in this experiment The established method was faster than the traditional methods which took several days.