Establishment Of Multiplex PCR For Staphylococcu Saureus, Streptococcus, Salmonella Spp And Escherichia Coli

Streptococcus, Staphylococcus aureus, Salmonella spp. and Escherichia coli widely distributed in nature and have serious consequences for livestock and poultry health, human public safety, food safety; and they are common pathogenic bacteria that cause a herd of pigs and human infect bacterial disease. The disease prevention and epidemiological investigation of Streptococcus,Staphylococcus aureus, Salmonella spp. and Escherichia coli, as well as to understand the food security situation and key problems of pathogens, all that dependent on timely and accurate detection and identification. In this study, four pairs of primers have been designed and synthesized according to highly conserved regions of the streptococcus ef-tu gene, staphylococcus aureus nuc gene, Salmonella spp hut gene and Escherichia coli 23SrRNA gene of NCBI. The multiplex PCR reaction condition was optimized and the multiplex PCR method for detecting the co-infection of Staphylococcus aureus, Streptococcus, Salmonella spp. and Escherichia coli was established.Through adjustment and optimization for the main factors of the multiplex PCR like annealing temperature and primer concentration, we finally determines that the best annealing temperature is 54℃, and best primer concentration is 0.4 pmol/μL. The specificity test showed that a fragment of 197 bp, 278 bp, 495 bp and 652 bp was amplified from genomic DNA of streptococcus, staphylococcus aureus, Salmonella spp. and Escherichia coli, respectively. No amplification was achieved from control groups of other bacteria. The sensitivity test showed that the multiplex PCR could detect genome DNA of 25.6 pg for Streptococcus, 33.2 pg for Staphylococcus aureus, 35.7 pg for Salmonella spp., and 52.1 pg for Escherichia coli, such sensitive far higher than the traditional bacteria separated and cultivated test methods.The artificial simulation test showed that the multiplex PCR established of this paper could detect four kinds of pathogens from co-infection disease material.The result indicated the multiplex PCR method had advantages of specificity, sensitivity, repetitive, and it could effectively detect the co-infection of streptococcus, staphylococcus aureus, Salmonella spp. and Escherichia coli. After further research and optimization, considering the multiplex PCR method can be applied in veterinary clinical diagnosis and food safety fast detection.