Detection of the gene encoding the subunit toxin B from Malaysian Vibrio cholerae isolates

Abstract

The gene encoding cholera toxin subunit B (ctxB) is the gene of focus in this study as the subunit toxin B is an effective immunogen. The ctxB was detected using polymerase chain reaction (PCR) based on the expected size (460 bp) in 20 isolates from local outbreaks of cholera in Peninsular Malaysia. These isolates consisted of Ogawa, Inaba and 0139 Bengal strains. The PCR conditions were optimised in different methods of template DNA preparations as well as primer and DNA concentrations. The PCR conditions were also specific against Escherichia coli, Klebsiella and Salmonella species. The identification of the ctxB PCR products was further confirmed using Southern hybridization and sequencing. For Southern hybridization, a 20-mer biotinylated oligonucleotide probe was used in detecting the PCR amplicon by a chemiluminescent detection method. The sequencing data obtained further confirmed that the amplicon is the gene of interest, and that the strain of V cholera used in this study belongs to the Classical biotype.