PCR detects hemolysin gene variation among Vibrio species not observed using DNA hybridization
Conejero, M.J.U. | Imbao, R.T. | Lising, J.S. | Hedreyda, C.T. (Philippines Univ. Diliman, Diliman, Quezon City (Philippines). National Inst. of Molecular Biology and Biotechnology)
Bacterial hemolysins are considered as important virulence factors of many Vibrio species, allowing them to cause disease in susceptible hosts. Hemolysin genes (vhhA and vhhB) have been cloned and isolated from a strain of Vibrio harveyi pathogenic to salmon. A set of primers designed based on hemolysin gene sequences from the Vibrio harveyi strain IFO 15634 was used to amplify the gene from different Vibrio species: V. parahaemolyticus, V. alginolyticus, V. proteolyticus, V. diazotrophicus, V. natriegens, V. vulnificus, V. campbellii and V. harveyi. Two bands representing amplified DNA fragments of about 210 and 1300 bp were obtained in PCR using DNA template from V. harveyi (IFO 15634). No PCR products were observed using DNA templates from other Vibrio species used. The 1300 bp amplified product was labeled and used in a non-radioactive hybridization experiment. Positive hybridization signals were observed, not only with the total genomic DNA of V. harveyi but also with the DNA from three other species, V. parahaemolyticus, V. alginolyticus, and V. proteolyticus. These results suggest that the other 3 Vibrio species possess a hemolysin gene with significant homology to the V. harveyi gene. However, if the PCR primers were designed to anneal to a relatively less conserved region of the gene, PCR detection could be specific for V. harveyi. Consequently, it may be possible to design primers that could be specific for the detection of hemolysin from other species as well. Therefore, hybridization could be used to detect if a homologous hemolysin gene is present in several species of Vibrio and PCR could detect hemolysin genes specific for a species
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