Study On The High Throughput Detection Technology Of Foodborne Pathogens By Tem-PCR And DHPLC

In order to solve imbalances of multiple PCR amplification and improve the detection efficiency and flux, we establish the high throughput detection methods of five food-borne pathogens. The method is simple, sensitive for staphylococcus aureus, salmonella bacteria hayes, and mononuclear cell hyperplasia liszt bacteria and e. coli bacterium 0157.According to the specific target gene sequences in GenBank of five pathogens, the complex primers were designed. Optimizing the concentration of specific primers by target sequence enrichment in multiplex PCR amplification, the best concentration was 80 nmol/ L.Staphylococcus aureus, Shigella and Salmonella bacteria were amplified by the three-step method of Tem-PCR amplification. The detection sensitivities were 2 x 103 CFU/mL,1.2 x 103 and 1.1 x 103CFU/mL. The result is similar with the tradition. It showed that the method possessed good detection specificity. Detecting the simulated and random food samples which were contaminated by Staphylococcus aureus Rosenbach, Shigella and Salmonella and contrast, it showed the method possessed good usability.The complex primers were designed of salmonella bacteria hayes, and mononuclear cell hyperplasia liszt bacteria and e. coli bacterium 0157. Five bacterium were applified by three to five applifications in Tem-PCR reaction system and conditions. The result showed that all applications were effective. There were absorption peaks which were detected by high-performance liquid chromatography. The detection sensitivity of Staphylococcus aureus Rosenbach was 620 cfu/mL. The others were less than 100 cfu/mL. It indicated that it possessed high detection sensitivities of five food-borne pathogens. It showed that the pathogens of ontaminated samples were detected by Tem-PCR-DHPLC method. And it was consonant with the national standard. It showed the good applicability.To compare with the traditional multiple PCR amplification, Tem-PCR with Tem-PCR-DHPLC detection method can effectively resolved the imbalances of application efficiency caused by primer concentration in PCR amplification. It needn’t optimization of primer concentration. It possessed good specificity, sensitivity and practicality.