Abstract
Sixty five isolates of Vibrio harveyi were subjected to random amplified polymorphic DNA (RAPD)-PCR analysis and protein profiling to investigate the genetic variability among V. harveyi prevalent along the coast and also assess the discriminating ability of these two molecular methods. A total of 10 RAPD primers were assayed for their specificity in detecting V. harveyi, of which only two primers: PM3 and CRA25 were highly reproducible and found suitable for use in RAPD-PCR. The genetic diversity among V. harveyi isolates assessed by RAPD-PCR using PM3 primer yielded 35 different RAPD patterns which clustered the isolates into 15 groups at 72% similarity level. Similarly, RAPD-PCR with CRA25 clustered the 38 patterns into 10 groups at 74% similarity. The discriminatory index (D) value calculated for RAPD fingerprints generated with PM3 and CRA25 were 0.90 and 0.85, respectively. On the other hand, molecular typing of V. harveyi using whole cell proteins generated profiles that showed no major difference indicating the technique to be not useful in typing strains of this bacterium. However, a few of the isolates showed the presence of unique band of 28 kDa that needs to be further investigated to understand the role of the protein in disease process if any.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Alavandi S. V., Manoranjita V., Vijayan K. K., Kalaimani N. and Santiago T. C. 2006 Phenotypic and molecular typing of Vibrio harveyi isolates and their pathogenicity to tiger shrimp larvae. J. Appl. Microbiol. 43, 566–570.
Ausubel F. M. 1995 Short protocols in molecular biology, 3rd edition. Wiley, New York, USA.
Conejero M. J. U. and Hedreyda C. T. 2003 Isolation of partial toxR gene of Vibrio harveyi and design of toxR-tergeted PCR primers for species detection. J. Appl. Microbiol. 95, 602–611.
Conejero M. J. U. and Hedreyda C. T. 2004 PCR detection of hemolysin (vhh) gene in Vibrio harveyi. J. Gen. Appl. Microbiol. 50, 137–142.
Dalla V. L., Zanella L., Belvedere P. and Colombo L. 2002 Use of random amplification to developed a PCR detection method for the causative agents of fish pasteurellosis. Photobacterium damselae subsp. piscicida (Vibrionaceae). Aquaculture 207, 187–202.
Dileep V., Kumar H. S., Kumar Y., Nishibuchi M., Karunasagar I. and Karunasagar I. 2003 Application of polymerase chain reaction for detection of Vibrio parahaemolyticus associated with tropical sea foods and coastal environment. Lett. Appl.Microbiol. 36, 423–427.
Harlow E. and Lane D. 1988 Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA.
Hernandez G. and Olmos J. 2004 Molecular identification of pathogenic and nonpathogenic strains of Vibrio harveyi using PCR and RAPD. Appl. Microbiol. Biotechnol. 63, 722–727.
Huey B. and Hall J. 1989 Hypervariable DNA fingerprinting in Escherichia coli: minisatellite probe from bacteriophage M13. J. Bacteriol. 171, 2528.
Hunter R. P. and Gaston A. M. 1988 Numerical index of the discriminatory ability of typing systems: an application of simpsons index of diversity. J. Clin. Microbiol. 6, 2465–2466.
Karunasagar I., Pai R., Malathi G. R. and Karunasagar I. 1994 Mass mortality of Penaeus monodon larvae due to antibiotic resistance Vibrio harveyi infection. Aquaculture 128, 203–209.
Krech T., de Chastonay J. and Falsen E. 1988 Epidemiology of diptheria: polypeptide and restriction enzyme analysis in comparison with conventional phage typing. Eur. J. Clin. Microbiol. Infect. Dis. 7, 232–237.
Lavilla-Pitogo C. R., Baticados M. C. L., Croz-Lacierda E. R. and Pena D. L. 1990 Occurrence of luminous bacterial disease of Penaeus monodon larvae in the Philippines. Aquaculture 91, 1–13.
Makemson J. C. and Hermosa Jr G. V. 1999 Luminous bacteria cultured from fish guts in the Gulf of Oman. Luminescence 14, 161–168.
Neilan B. A. 1995 Identification and phylogenetic analysis of toxigenic cyanobacteria by multiplex randomly amplified polymorphic DNA PCR. Appl. Environ. Microbiol. 61, 2286–2291.
Oakey H. J., Levy N., Bourne D. G., Cullen B. and Thomas A. 2003 The use of PCR to aid in the rapid identification of Vibrio harveyi isolates. J. Appl. Microbiol. 95, 1293–1303.
Pizzutto M. and Hirst R. G. 1995 Classification of isolates of Vibrio harveyi virulent to penaeus monodon larvae by protein profile analysis and M13 DNA fingerprinting. Dis. Aquat. Org. 21, 61–68.
Pujalte M. J., Sitjà-Bobadilla A., Macián M. C., Belloch C., Álvarez-Pellitero P., Perez-sanchez J. et al. 2003 Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex, gilthead sea bream and European sea bass. Sys. Appl. Microbiol. 26, 284–292.
Shangkuan Y. H., Tsao C. M. and Lin H. C. 1997 Comparison of Vibrio cholerae O1 isolates by polymerase chain reaction fingerprinting and ribotyping. J. Med. Microbiol. 46, 941–948.
Somarny W. M. Z., Mariana N. S., Neela V., Rozita R. and Raha A. R. 2002 Differentiation of pathogenic Vibrio species by RAPD. J. Med. Sci. 2, 165–169.
Tassanakajon A., Pongsomboon S., Rimphanitchayakil V., Jarayabhaud P. and Boonsaeug V. 1997 Random amplified polymorphic DNA (RAPD) markers for determination of genetic variation in wild population of the black tiger prawn (Penaeus monodon) in Thailand. Mol. Mar. Biol. Biotechnol. 6, 110–115.
Welsh J. and McClelland M. 1993 The characterization of pathogenic micro-organisms by genomic fingerprinting using arbitrarily primed polymerase chain reaction. (AP-PCR). In Diagnostic molecular microbiology (ed. D. H. Persing, T. F. Smith, F. C. Tenover and T. J. White), pp. 595–602. ASM Press, Washington DC, USA.
West P. A. and Colwell R. R. 1984 Identification and classification of Vibrionaceae — an overview. In Vibrios in the environment (ed. R. R. Colwell), pp. 285–363. John Wiley, New York, USA.
Williams J. G. K., Kubelik A. R., Livak K. J., Rafalski L. A. and Tingey S. V. 1990 DNA polymorphisms amplified by arbitrary primers are useful as genetic marker. Nucleic Acids Res. 18, 6531–6535.
Yeh F. C. and Boyle T. J. B. 1997 Population genetic analysis of co-dominant and dominant markers and quantitative traits. Belg. J. Bot. 129, 157.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Maiti, B., Shekar, M., Khushiramani, R. et al. Evaluation of RAPD-PCR and protein profile analysis to differentiate Vibrio harveyi strains prevalent along the southwest coast of India. J Genet 88, 273–279 (2009). https://doi.org/10.1007/s12041-009-0040-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12041-009-0040-z