Rapid Detection of Salmonella enteritidis by PCR Amplification of The SefA Gene and It`s Cloning

Abstract

The emergence of Salmonella enteritidis as an important food-borne pathogenesis in humans, demands the development of novel detection and intervention strategies. It is generally accepted that fimbriae are an important factor in bacterial survival and persistence in the host. This study is directed towards the method of amplifying and cloning the SefA gene, which encode Salmonella enteritidis fimbrial protein. Strains used for these studies were S. enteritidis (E3), which were collected from Kermanshah region. Chromosomal DNA was extracted by boiling method and PCR reaction was performed and single band of 511 bp amplified by SefA-F and SefA-R primers. The resulting PCR product was inserted into the cloning vector (pTZ57R/T). In order to amplify the recombinant plasmid, E. coli DH5 alpha bacteria were transformed with SefA-pTZ57R/T. Recombinant clones were identified by blue/white selection and purified recombinant plasmids were indicated by an alkaline lysis procedure. Identity of the SefA-pTZ57R/T product was confirmed by RFLP and sequencing. Nucleotide and protein alignment with BLAST software showed that the sequence of the SefA gene derived from S. enteritidis (E3), which was cloned in the pTZ57R/T vector, was 99% identical to that of the Genbank (L11008). The sequence of the SefA gene from S. enteritidis (E3) differed only in two nucleotides and one amino acid. The cloned SefA gene from S. enteritidis (E3) was submitted to the NCBI Genbank (EF553334).