title: GroEL PCR- RFLP - An efficient tool to discriminate closely related pathogenic Vibrio species.
abstract: Vibrio sp. are autochthonous to marine and estuarine waters. Several species of Vibrio are pathogens. It is of utmost importance to detect and discriminate the Vibrio sp. that are often involved in food and water borne infections. Since 16S rRNA based identification has limited utility in differentiating the closely related pathogenic species from non pathogenic species, we have evaluated the discriminatory power of groEL PCR-RFLP for identification of closely related Vibrio sp. Accordingly, in the current study, the efficiency of groEL PCR- RFLP for detection and accurate differentiation of known pathogens among Vibrio sp. such as V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. fluvialis, V. alginolyticus, V. anguillarum was evaluated. PCR amplified groEL gene fragment of each Vibrio sp. was digested separately using 5 restriction enzymes viz. Hha1, Rsa1, Alu1, Dde1 and Mbo1. The accuracy of the method was further validated by insilico restriction analysis of multiple strains of each species using NEBcutter. The method proved to be efficient for detection and differentiation of Vibrio species under study. Phylogenetic analysis also revealed groEL gene to be a better phylogenetic marker for Vibrio compared to 16S rRNA. Hence, the method can be employed for accurate detection of Vibrio sp. including the closely related species.
authors.affiliation: Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Lakeside Campus, Cochin University of Science and Technology, Cochin 682016, India. Electronic address: sanasilvester@gmail.com.
authors.forename: Reshma
authors.initials: R
authors.lastname: Silvester
authors.affiliation: Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Lakeside Campus, Cochin University of Science and Technology, Cochin 682016, India.
authors.forename: Deborah
authors.initials: D
authors.lastname: Alexander
authors.affiliation: Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Lakeside Campus, Cochin University of Science and Technology, Cochin 682016, India.
authors.forename: Ally C
authors.initials: AC
authors.lastname: Antony
authors.affiliation: Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Lakeside Campus, Cochin University of Science and Technology, Cochin 682016, India.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Hatha
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D018834:Chaperonin 60 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D003198:Computer Simulation; D005798:Genes, Bacterial; D008828:Microbiological Techniques / Q000379:methods; D010802:Phylogeny; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D014733:Vibrio / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification / Q000472:pathogenicity
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D012336:RNA, Ribosomal, 16S
keywords: Detection; GroEL gene; PCR- RFLP; Pathogens; Vibrio sp.