OPTIMIZATION OF THE ARBITRARILY-PRIMED POLYMERASE-CHAIN-REACTION (AP-PCR) FOR INTRASPECIES DIFFERENTIATION OF VIBRIO-VULNIFICUS

Optimal parameters were established for obtaining unique and reproduci ble DNA fingerprints of selected clinical and environmental isolates o f Vibrio vulnificus by the arbitrarily primed polymerase chain reactio n (AP-PCR). Genomic DNA from selected strains was subjected to AP-PCR amplification using a single, arbitrarily selected oligonucleotide pri mer, R-PSE420. Amplified DNA was analyzed by agarose gel electrophores is and ProRFLP(R) computer software. Reproducibility of the fingerprin t was dependent upon the concentrations of the purified genomic DNA, M gCl2 and oligonucleotide primer, and on PCR cycling parameters. Using 1 mu g of purified genomic DNA, 2.5 mM MgCl2, 1.04 mu M of R-PSE420 ol igonucleotide primer and thermal cycling protocols with stepwise incre ases in the annealing temperatures, DNA fingerprints which were reprod ucible and free of primer artifacts were generated. By following the o ptimized AP-PCR amplification protocol, unique DNA fingerprint profile s for each V. vulnificus strain tested were produced. These AP-PCR gen erated unique DNA fingerprint profiles can be used in the identificati on, and investigation of the distribution and diversity of various str ains of V. vulnificus in bivalve shellfish and their surrounding water s. (C) 1998 Elsevier Science B.V.