Study On Detection Of Food-borne Pathogenic Bacteria By Multiplex PCR

Food is the god for the people. Food safety is the first for people. Food safety emergeneies are high frequency in recent years. Food contamination with bacterial pathogens are major reasons of food-borne disease. Strengthen the food inspection efforts is the most effective way to solve the problem of food safety. At present, detection of food-borne pathogens mainly uses the traditional isolation culture and biochemical identification and PCR methods in our country. The 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 development 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 approach would be relatively rapid and cost-effective. Multiplex PCR which can simultaneously detect more than one bacterium has been applied increasingly comprehensive.To develope a rapid, sensitive and specific method for detection of Listeria monocyto, Staphylococcus aureus and Escherichia coli O157∶H7 by multiplexes PCR at the same time. Frist according to transcriptional regulatory protein (hlyA ) gene of Listeria monocyto, the heat-stable nuclease (nuc) gene of Staphylococcus aureus and O157 antigen (rfbE)gene of enterohemorrhagic E. coli O157:H7, three pairs of specific primers were designed by Primer 5.0 for multiplex PCR amplification. The multiplex PCR system were optimized to make three kinds of food-borne pathogens have better amplification results. Multiplexes PCR products were examined by 1.5% agarose gel electrorophresis. Moreover the amplification fragments were analyzed by BLAST to avoid high homology and complementarity through GeneBank .In this study, Specificity of each simplex multiplex PCR assay was validated by testing various bacteria including positive isolates and negative isolates. The results showed that three pairs of primers produced specific amplicons of expected sizes 439 bp for Listeria monocyto, 230 bp for Staphylococcus aureus , 151 bp for Escherichia coli O157∶H7. The specificity of the multiplex PCR assay was evaluated by testing the four primer sets with the purified DNAs of all the strains (separately or in different combinations) indicated above. Positive PCR amplifieation of DNA templates from Listeria monocyto, Staphylococcus aureus and Escherichia coli O157∶H7. Produced a single fragment of the expected for each Pathogen. The four baeterial pathogens were simultaneously amplified.The multiplex PCR amplification conditions were optimized. In the multiplex PCR process Mg2 +concentration, primer concentration,template volume,annealing times and temoerature are optimized to determine the optimal PCR. The reaction mixture consisted of 50μL: 5μL 10×PCR buffer, 3.5μL Mg2+(25 mM), 5.5μL mixture of dNTPs,0.6μL(5 U/μL)Taq,2μL of 10μM primer HlyA(each),2.2μL of 10μM primer nuc(each), 1.8μL of 10μM primer rfbE(each), 2μL of DNA(each), 17.4μL ddH2O. The reaction was run under the following conditions: Cool start; DNA pre-denaturation at 94℃for 5 min, DNA denaturation at 94℃for 1 min, primer annealing at 55.4℃for 45 s, and DNA extention at 72℃for 1 min, run 33 cycles; the final extention was performed at 72℃for 5 min.In this study , The sensitivity of the multiplex PCR for the detection one of three bacterial pathogens when using purified DNA of the bacterial type strains was 101 CFU/mL for Listeria monocyto, Staphylococcus aureus and Escherichia coli O157∶H7. At the same time, The sensitivity of the simultaneous detection of the three bacterial pathogens with the multiplex PCR when using purified DNA of the bacterial type strains was 102 CFU /mL, The detection limit of the multiplex PCR assay for food detection was 103 CFU /mL.Compared with the national standard method, The sensiticity of the multiple PCR assay is 100%, The specificity is 95.6% for Listeria monocyto, 92.3% for Staphylococcus aureus, 92.8% for Escherichia coli O157∶H7, The coincidence rate is 96.7% for Listeria monocyto, 93.3% for Staphylococcus aureus, 96.7% for Escherichia coli O157∶H7. The whole experimental procedure can be completed only 6 hours, shorter than the traditional biochemistry detection. The results show that the multiplex PCR detection method is simple, rapid, strong specificity, high sensitivity and has good application prospect.