Establishment And Application Of Multiplex PCR For Detection Of Three Food-borne Bacterial Pathogens

In recent years, global food safety incidents are in high frequency and become a vital factor affecting public health. In these problems, the food poisoning result of bacterial pathogens was the most harmful. Thus, food pathogenic bacterias detection is an important aspect in food hygiene and safety testing. 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 foodborne microbiological techniques often require several days to detect bacterial pathogens present in foods at low levels, and can only detects one kind of bacterial each time. Face of increasing multiplex mixed pollution, the current detection method appears to lag. Therefore, the establishment of efficient, rapid and accurate the detection methods of food pathogenic bacterias become more and more urgent. In recent years, PCR technology is developing very rapidly. Multiplex PCR technology can achieve in detecting a variety of pathogenic microorganism in a tube, with the advantage of high efficiency, low cost and high sensitivity. Therefore, multiplex PCR assays were developed for the simultaneous detection of Staphylococcus aureus, Salmonella spp. and the Bacillus cereus in this study.In this study, took use of Staphylococcus aureus of special sequences of conservative nuc gene, Salmonella spp. invA gene and Bacillus cereus hblA gene to design three special primers. In the m-PCR process, annealing temperature, Mg2+ concentration, dNTPs and PCR circles are optimized to determine the optimal m-PCR. The reaction mixture consisted of 25μL: 2.5μL10×PCR buffer, 2.0μL MgSO4, 2.0μL dNTP Mixture, 1μL of 10μM forward primer(each), 1μL of 10μM reverse primer(each), 0.5μL Taq enzyme(5 U), 2μL template(each), ddH2O 7μL. The reaction was run under the following conditions: Cool start, DNA pre-denaturation at 94°C for 5 min; DNA denaturation at 94°C for 40 s, Primer annealing at 59°C for 40 s, and DNA extending at 72°C for 40 s, run 30 cycles; the final extending was performed at 72°C for 10 min.There were 22 bacterial strains to be detected including 4 Staphylococcus aureus strains, 4 Salmonella spp. and 4 Bacillus cereus and 10 other bacterial strains in order to determine specificity of amplification of primers. The results of 4 Staphylococcus aureus strains, 4 Salmonella spp. and 4 Bacillus cereus were positive and those of 10 other strains were negative. The specificity of the m-PCR assay was evaluated by testing the three primers set with the purified DNAs of all the strains (separately or in different combinations) indicated above. Moreover the primers and amplification fragments were analyzed by BLAST to confirm high homology and complementarity through GeneBank. It is confirmed that their homologies are the same at 99%.The sensitivity of m-PCR for the detection of one of three bacterial pathogens when using purified DNA of the bacterial type strains was l03 CFU/mL for Staphylococcus aureus and Bacillus cereus, l04 CFU/mL for Salmonella spp. The sensitivity of the simultaneous detection of the three bacterial pathogens with m-PCR when using purified DNA of the bacterial type strains was l04 CFU/mL.This study compared five different methods extract DNA samples from template to amplificate by m-PCR, when the pure milk was polluted by three pathogenic bacteria concentrated. In the results, the method of solvent extraction method of thermal cracking was used to amplificate by m-PCR are more stable, easy operation, and low price. The bacterial pathogens’DNA was directly extracted by using solvent extraction method of thermal cracking. The detection limit of the m-PCR assay for milk was l02 CFU/mL for Staphylococcus aureus and Bacillus cereus, l04 CFU/mL for Salmonella spp. The detection limit of the simultaneous detection of the three bacterial pathogens in milk with m-PCR was l04 CFU/mL.In this study, real detection of 50 samples was made. The result indicates: the sensitivity of the m-PCR assay is 100%. The specificity is 93.5% for Staphylococcus aureus. 94.7% for Salmonella spp., 90.6% for Bacillus cereus. The coincidence rate is 96% for Staphylococcus aureus, 92% for Salmonella spp., 94% for Bacillus cereus.Multiplex PCR assay for the simultaneous detection of Staphylococcus aureus, Salmonella spp. and Bacillus cereus have been established in this study. The established multiplex PCR method whose inspection time was about 6 h had the advantages of sensitive, specific, accurate and rapid. Therefore, the multiplex PCR detection method has practical value in basic level, with important significance in detecting foodborne pathogenic bacteria timely, reducing food poisoning accidents and guaranteeing the national food safety.