| In recent years,global food safety incidents are in high frequency and become a vital factor affecting public health.food safty problems has increasingly become a focus of social concern. Microbial food poisoning is in first place among the foodborne diseases prevalent in our country. Thus, food pathogenic bacterias detection is an important aspect in food hygiene and safety testing. At present.Detection of foodborne pathogens mainly uses the traditional isolation, cμlture and biochemical identification method, which is complicated operation. Time-consuming, and can only detects one kind of bacterial each time. It usually takes about one week in order to submit test reports, and the sensitivity and specificity are low. Face of increasing multiple mixed pollution, the current detection method appears to lag, Therefore the establishment of efficient, rapid and accurate detection of foodborne pathogenic microorganisms methods become more and more urgent.Multiplex PCR has the same basic principle with conventional PCR, a number of primers is added to same PCR reaction system,achieve more than one target genes amplification in the same system at the same time, it overcomes the drawback that conventional PCR reaction can only amplify one target gene. Multiplex PCR provides a wider space for development, so as to achieve the fast detection of a variety of foodborne pathogenic bacterias at the same time, saving cost and time.To establish a PCR detection method of the four pathogens at the same time. In this study, select the IpaH gene of Shigella, hlyA gene of Listeria monocytogenes bacteria, nuc gene of Staphylococcus aureus, the hlyA gene of E. coli 0157:H7 as target genes. design and select four paris of specific primers, the length of target gene fragment was 113 bp,155bp,283bp,366 bp through PCR amplification. First, create a single PCR detection system to amplify the standard strains and determine the detection specificity and sensitivity. sequencing shows their identity up to 99%. of good specificity,sensitivity of 101cfu/ml. Thus the single PCR detection system Lays the foundation for the establishment multiplex PCR detection system.Establish multiplex PCR detection system. According to the main influnce factors of multiplex PCR, such as primer concentration. dNTP concentration. Taq enzyme concentration and Mg2+ concentration. Design L9 (34) orthogonal experiment to find the optimal reaction conditions, then optimize annealing temperature.Final reaction system is 50μl. Also established a two-step PCR amplification system and circμlatory conditions. The results showed that all of 4 strains of bacteria have clear, specific goals bands. Sensitivity of Shigella and Listeria monocytogenes reach to 101cfu/ml.0157 and Staphylococcus aureus to the 102cfu/ml. therefore, in this study,sensitivity of mμltiple PCR system for detection of the four pathogens reached to 1.8 x 105cfu/ml.At the same time, the national standard method as control, human milk samples inoculated with bacteria were detected.also 84 Commercial products such as Instant noodles, tea, sweetened bean paste, stick frozen corn, frozen rabbit meat, chicken, frozen boiled crab were detected, the samples are cultured by LB before detected. The results showed that multiple PCR method is simple, fast and high sensitivity, the detection time in less than 24 h, Compared with the national standard, Multiplex PCR detection of the 84 samples is no false negative and false positive less than 2%, The multiplex PCR method is sensitive, specific, can achieve rapid detection of Shigella, Staphylococcus aureus, enterohemorrhagic Escherichia coli 0157:H7, Lisi monocytogenes strains in food samples, it has broad prospects in pathogen detection.at the same time,it will play a imporant part in controling pathogen transmission, preventing the occurrence of food poisoning in future.
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