Optimization of the arbitrarily-primed polymerase chain reaction (AP-PCR) for intra-species differentiation of Vibrio vulnificus

Abstract

Optimal parameters were established for obtaining unique and reproducible DNA fingerprints of selected clinical and environmental isolates of Vibrio vulnificus by the arbitrarily primed polymerase chain reaction (AP-PCR). Genomic DNA from selected strains was subjected to AP-PCR amplification using a single, arbitrarily selected oligonucleotide primer, R-PSE420. Amplified DNA was analyzed by agarose gel electrophoresis and ProRFLP® computer software. Reproducibility of the fingerprint was dependent upon the concentrations of the purified genomic DNA, MgCl 2 and oligonucleotide primer, and on PCR cycling parameters. Using 1 μg of purified genomic DNA, 2.5 mM MgCl 2, 1.04 μM of R-PSE420 oligonucleotide primer and thermal cycling protocols with stepwise increases in the annealing temperatures, DNA fingerprints which were reproducible and free of primer artifacts were generated. By following the optimized AP-PCR amplification protocol, unique DNA fingerprint profiles for each V. vulnificus strain tested were produced. These AP-PCR generated unique DNA fingerprint profiles can be used in the identification, and investigation of the distribution and diversity of various strains of V. vulnificus in bivalve shellfish and their surrounding waters.