Detection Of Salmonella, Shigellae And Staphylococcus Aureu By Multiplex Real-time PCR Based On SYBR Green â… 

Salmonella, Shigellae and Staphylococcus aureu are pathogeny bacterium that can induce the food poisoning. They are importance detect item in food security risk assessment, they also are necessarily detect target of pathogeny bacterium at food detect standard like soy sauce, freeze drink, meat product, egg products, dairy produce etc. in China. To establish a multiplex single-tube real-time PCR approach for the detection of Salmonella, Shigellae and Staphylococcus aureu, it will be able to fulfill the actual detection requirements, shorten the time and improve efficiency of pathogen detection.The study according to the characteristics that SYBR Green I can integrate with double stranded DNA, establish a multiplex real-time PCR approach for the detection of Salmonella, Shigellae and Staphylococcus aureu. It include two parts:1.A Enrichment Broth for Simultaneous Growth of Salmonella, Shigellae and Staphylococcus aureuAccording to the six micro-nutrient requirement, compare the different component and fix on the component of enrichment medium, develop a enrichment broth for simultaneous growth of Salmonella, Shigellae and Staphylococcus aureu. When 10 CFU per one milliliter of Salmonella, Shigellae and Staphylococcus aureu were simultaneously added, and incubated for 12h at 37℃, multiplied at nearly same speed,and reached at 107～108 CFU per one milliliter,multiplex PCR can be used to produced specific amplicons of expected sizes. The enrichment broth confecting convenient, increasing bacterial rapidly, saving cost and time.2. Establish a multiplex real-time PCR approach for the detection of Salmonella, Shigellae and Staphylococcus aureuThe study select the Salmonella. invA gene, Shigella ipaH gene and virA gene, Staphylococcus aureus nuc gene and femA gene for targets then design several pairs of specific primers. We compare Tm values to fix the three pairs of primers and optimize the reaction conditions to establish the multiplex real-time PCR approach for the detection of Salmonella, Shigellae and Staphylococcus aureu. The sensitive of the multiplex real-time PCR assay was 1.68×102CFU/mL,1.36 X 102CFU/mL and 1.24 X 103CFU/mL. The multiplex:real-time PCR assay showed good specificity which was validated by testing various bacteria isolates. It could directly detect the three pathogenic bacteria simultaneously, the detection limit was 102CFU/ g(mL). The detection sensitivity in meat reached 1 CFU/ g(mL) after 12h enriched culture. The multiplex real-time PCR assay is accurate, high efficient and is valuable for food sanitation detection, clinical inspection field and widely used.