Rapid detection of Salmonella dublin by PCR amplification of the SopE gene and its cloning.
This study is directed towards the method of amplifying and cloning the SopE gene, that encodes Salmonella outer protein E. Strains used in this study were S. dublin collected from Kermanshah province. Genomic DNA was extracted by the general boiling method. Using the specific primers, a part of SopE gene was multiplied. The PCR product was inserted into the cloning vector (pTZ57R/T). Furthermore, E. coli DH5alpha bacteria were transformed to amplify the recombinant plasmid. Recombinant clones were identified by blue/white selection. Recombinant plasmids were purified by alkaline lysis procedure. Moreover, identity of the SopE/pTZ57R/T product was confirmed by restriction enzyme digestion assay and sequencing. Finally, the cloned gene was compared with that published by the NCBI Genbank (L78932). The results showed that the obtained sequence differed in four nucleotides which resulted in two amino acid differences. The cloned SopE was submitted to the NCBI Genbank (EU399750).
Mirmomeni
M H
MH
Cell and Molecular Unit, Department of Biology, Razi University, Kermanshah, Iran.
Kiani
S
S
Sisakhtnezhad
S
S
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004587:Electrophoresis, Agar Gel; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D017931:DNA Primers; C103987:SopE protein, Salmonella