| Food safety emergencies are high frequency in recent years. In these problems, the food poisoning result of Bacterial Pathogens was the most harmful. These conventional food microbiological techniques often require three to five days to detect bacterial pathogens present in foods at low levels. Therefore, it is urgent to set up a kind of fast effective and flux higher detection methods.Three pairs of primers were designed respectively according to the invasive protein gene invA of Salmonella, the nuc gene of Staphylococcus aureus, the sequence of t he ail gene of Yersinia enterocolitica. In the process of test of multiple reaction system, annealing temperature, primers addition, Mg2+ addition, dNTPs addition and Taq enzymes addition are optimized to determine the optimal PCR. The specificity of primers, determined the sensitivity, and explored the extracted from food three pathogenic bacteria template DNA method, inspection artificial pollution samples, determined the detection limit, and with the traditional test methods were compared.The reaction mixture consisted of 25μL, 10×PCR buffer 2μL, Mg2+(25mmol/L)1μL, 0.8μL mixture of dNTPs(25 mM), Taq enzyme(5U/μL)0.3μL, template 2μL, 0.8μL of forward primer(each), 0.8μL reversse primer(each), ddH20 to 25μL. The reaction was run under the following conditions: DNA pre-denaturation at 94℃for 5 min, DNA denaturation at 94℃for 45 s, primer annealing at 57℃for 30 s, and DNA extention at 70℃for 50s, run 30 cycles; the final extention was performed at 72℃for 10 min. The PCR Products were examined by 2% agarose gel electrophoresis. Three DNA fragments of 475, 236 and 127 bp were amplified by three pairs of specific primers. PCR products were confirmed by DNA sequencing. The results of sequence compared with the target gene sequence at gene bank showed that PCR amplified products were certified.This study detected in all 18 strains of bacteria, including 4 salmonella strains, 1 Staphylococcus aureus strains and Yersinia enterocolitica strains were all positive and those of 12 other strains were negative. The primers specificity was high. Determined the sensitivity, the results showed: the sensitivity of m-PCR detection one of three of bacterial pathogens, Salmonella was 102 CFU/mL, Staphylococcus aureus was 103 CFU/mL and Yersinia enterocolitica was 103 CFU/mL. The sensitivity of this simultaneous detection of the three bacterial pathogens with m-PCR when using purified DNA of the bacterial type strains was 103 CFU/mL for Salmonella, 103 CFU/mL for Staphylococcus aureus and 103 CFU/mL for Yersinia enterocolitica.The effect of 6 methods of extracting DNA from three bacterial pathogens in milk was compared, the result indicates a quick, effective and appropriate extracting DNA method was selected.Detection artificial pollution samples, the detection limit of the m-PCR assay for pasteurized milk was 102 CFU/mL for salmonella, 102 CFU/mL for Staphylococcus aureus, and 102 CFU/mL for Yersinia enterocolitica. The whole experimental procedure can be completed only 6 hours, shorter than the traditional biochemistry detection. In this study, real detection was made. The result indicates: the sensitivity of the m-PCR assay is 100%; the specificity is 95.5% for salmonella, 92.6% for Staphylococcus aureus, and 97.4% for Yersinia enterocolitica; the coincidence rate is 96.0% for salmonella, 96.0% for Staphylococcus aureus, 98.0% for Yersinia enterocolitica.We thus present a rapid method with both high sensitivity and specificity for the immediate detection and identification of three specific pathogenic bacteria and the m-PCR the must popularize value was the m-PCR. |
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