Research The Detection Method Of Fourborne Pathogenic Bacteria By PCR

Food safety emergencies are high frequency in recent years. In these problems,the food poisoning result of Bacterial Pathogens was the most harmful. According to data came from hospitals and inspection-quarantine departments,the main bacterial pathogens were Staphylococcus aureus, Salmonella spp, listeria monocytogenes and so on. Since contamination levels are generally low in foods, detection methods require lengthy culture enrichment steps to increase target bacterial numbers before isolation and identification using standard cultural procedures. These conventional food microbiological techniques often require several days to detect bacterial pathogens present in foods at low levels. With the advent of PCR, Individual PCR assays have been developed for detection and identification of the bacterial pathogens. but a large number of individual PCR assays would be necessary if single primer sets are used in separate reactions on a large number of food samples, which can be a relatively costly and time-consuming process. To reduce the number of tests needed for diagnosis of Individual PCR assays,the simultaneous detection of several pathogens with a multiplex PCR (m-PCR) approach would be relatively rapid and cost-effective.For establishing a method of diagnosing four species common animal-borne pathogenic bacteria simultaneously by multiplex PCR. Four pairs of specific Primers were designed according to the gene 16s-23s rRNA of Escherichia coli , the gene nuc of Staphylococcus aureus,the gene hlyA of Listeria monocytogenes and the gene invA of Salmonella spp . The target gene fragment amplified by PCR respectively was 662 bp , 484 bp , 372 bp , 284 bp , In the PCR process, DNA concentration, Mg²⁺ concentration, template volume, annealing temperature and PCR circles are optimized to determine the optimal PCR. The reaction mixture consisted of 50μL,10×PCR buffer 5μL, Mg²⁺(25mM)3μL, 4μL mixture of dNTPs, Taq enzyme(2.5 U)1μL, template 2μL,ddH20 to 50μL .The reaction was run under the following conditions: Cool start; DNA pre-denaturation at 94℃for 5 min, DNA denaturation at 94℃for 45 s, primer annealing at 57℃for 1 min ,and DNA extention at 70℃for 1 min30s, run 32 cycles; the final extention was performed at 70℃for 20 min.The established multiplex PCR method whose inspection time was about 5h had the advantages of sensitive , specific , accurate and rapid. It lays the foundation for researching and developing a kit that detects the main food-home pathogenic bacterium of zoonosis simultaneously , that is Escherichia coli , Staphylococcus aureus , Listeria monocytogenes and Salmonella spp.