Study On Polymerase Chain Reaction Molecular Detection Of Five Food-borne Bacterial Pathogens In Polluted Water

Food-borne bacterial pathogens were defined to be pathogens that could lead to diseases through food. The steps of conventional culture methods and biochemical confirmation were trivial, and need many days to acquire the result. The technique of immunology had the problems of specificity and sensitivity. We designed the specific primers, then developed the monoplex PCR and multiplex PCR detection techniques for detecting five kinds of bacterial pathoges which were Salmonella sp. Shigella sp., Pseudomonas aeruginosa, Eterohaemorrhagic O157 and Vibrio rahaemolyticus in water.Through optimized the system and condition of monoplex-PCR and multiplex-PCR, the detection system and identification condition of pathogenic bacteria in water not only be evaluated on DNA templates of thirty-five different bacterial isolates from ten species, but also can be applied to differentiate bacteria in manual polluted samples, we got the right and predicted electrophoretic result, and the comparative result of the monologue among five primer pairs were coincident. So the five pairs of oligonucleotide primers which were targeted at species-specific region and virulence-associated genes. It took five to six days for a sample detection.The sensitivity of monoplex PCR were:10~0 cfu/mL, 10~0cfu/mL, 10~-1cfu/mL, 10~0cfu/mL and 10~-1cfu/mL for Eterohaemorrhagic 0157, Shigella sp, Vibrio rahaemolyticus, Pseudomonas aeruginosa and Salmonella sp, respectively. The sensitivity of multiplex PCR were:10~1cfu/mL, 10~2cfu/mL, 10~2cfu/mL, 10~2cfu/mL and 10~1cfu/mL for Eterohaemorrhagic 0157, Shigella sp, Vibrio rahaemolyticus, Pseudomonas aeruginosa and Salmonella sp, respectively. The method established shortened the detection time and provided a cost-effective way in maximum. Besides, we had constituted the detection kits of monoplex PCR and multiplex PCR under the base of PCR methods. They would be generalized in those areas which were environmental inspection, water detection, food sanitation supervision , commodity inspection and detection, etc.