Detection of Salmonella, Shigellae and Staphylococcus Aureu by Multiplex Real-time PCR Based on SYBR Green Ⅰ
|School||Jilin Agricultural University|
|Course||Of Food Science|
|Keywords||Salmonella Shigella Staphylococcus aureus Enrichment medium Multiplex fluorescence quantitative PCR|
Salmonella (Salmonella), of Shigella (Shigellae), Staphylococcus aureus (Staphylococcus aureus) is the main pathogen causing food poisoning, food safety risk prediction and hazard assessment monitoring project of soy sauce, sauce, frozen drinks detection of pathogens in cooked meat, egg products, dairy products and other food testing standards will be seized indicators. To establish a rapid and accurate detection methods for these three pathogens, can meet the actual detection requires, the conventional method could be improved long existence Processed operation cumbersome insufficient. In this study, based on the SYBR Green I and double-stranded DNA binding characteristics, the use of quantitative PCR technique to optimize establish three pathogens in multiple fluorescence quantitative PCR system for Salmonella, Shigella and Staphylococcus aureus main research two parts: 1. simultaneously enriched Salmonella, Shigella and Staphylococcus aureus enrichment medium for microbial nutrient needs, screening can add ingredients used for univariate comparison test to determine the medium appropriate components obtained a way to for Salmonella, Shigella and Staphylococcus aureus composite enrichment medium when inoculated with approximately 10 CFU / mL, 37 ° C cultured 12h, three target bacteria proliferation of relatively consistent speed, up to 107 ~ 108 CFU / mL, three purposes simultaneously amplified by multiplex PCR bands. The composite enrichment medium for convenient preparation, enrichment fast, cost and time savings. Salmonella, Shigella and Staphylococcus aureus establishment of multiple fluorescence quantitative PCR method to select the Salmonella invA gene, the of Shigella ipaH genes and virA genes, Staphylococcus aureus Nuc gene and femA gene, design and more specific primers, comparative analysis of Tm value differences identified three pairs of specific primers, and reaction conditions were optimized, Salmonella, Shigella and Staphylococcus aureus multiple real-time fluorescence quantitative PCR. The sensitivity of detection of target bacteria bacterium DNA respectively 1.68X 102CFU/mL of from 1.36 X 102CFU/mL and 1.24 × 103CFU/mL direct detection of the lowest limit of detection for the mock-infected bacteria samples 102CFU / g (mL), detected after 12h growth analog samples of the bacteria, the detection limit was 1CFU / g (mL). Selected target strains and the strains of non-target specificity experiments, multiple quantitative PCR system has good specificity. The results show that established in this study, SYBR Green I multiplex fluorescence quantitative PCR specificity, sensitivity and reproducibility are up to the requirements of the experimental design, to provide the technical means for the detection of pathogenic bacteria, with a wide range of application value.