PCR Detection of Salmonella Typhimurium using Hin GENE

Salmonella cause disease in a wide range of species of vertebrates. The knowledge of the serotypes helps to define the sources and vehicles of infection in outbreaks of food poisoning. The isolated genomic DNA from Salmonella typhimurium was amplified with the designed oligonucleotide primers for the amplification of Hin gene. The genomic DNA was serially diluted and the least expressed concentration of each genomic DNA for amplification was selected and performed the multiplex PCR, to detect the presence of Hin gene thereby detecting Salmonella typhimurium. The genomic DNA from bacterial cells was recovered using the salting out method. Oligonucleotide primers were designed for PCR amplification of Salmonella spp. Primer annealing temperatures were calculated by oligo version computer programme. PCR amplification was performed with a model Gene Amp PCR system 9700(Perkin Elmer) by using 1X PCR buffer ,2.5mM MgCl , deoxynucleoside triphosphate at a concentration of 200μM.primer at a concentration of 0.5 2 M,100ng of target DNA and 2.5U of DNA Polymerase . PCR amplified DNA was analyzed by gel electrophoresis. At an annealing temperature of 550C a specific amplification was obtained with a size of 230bp for Hin gene, 200bp for Salmonelli abony,450 bp for Pseudomonas aeruginosa and 500bp for E.coli. The size of the PCR product was confirmed by comparing with a marker of 100bp which also stands true for the product size obtained by doing NCBI BLAST. The blast results suggested the amplifies sequence is showing 99% homology to the S. typhimurium.