APPLICATION OF RANDOMLY AMPLIFIED POLYMORPHIC DNA-FINGERPRINTING FOR SPECIES IDENTIFICATION OF BACTERIA ISOLATED FROM BOVINE-MILK

A polymerase chain reaction-based DNA fingerprinting system for specie s identification of bacteria in milk was developed using randomly ampl ified polymorphic DNA. A total of 108 organisms including 24 ATCC refe rence strains and 84 wild-type isolates belonging to gram-negative, St aphylococcus, Enterococcus, and Streptococcus species were used to dev elop the system. Organisms included in the study were those that are i solated frequently from milk. Forty primers fi om two commercially ava ilable primer kits were evaluated to determine the ''ideal'' primer th at could be used for several bacterial species. Over 960 DNA fingerpri nt patterns were analyzed by laser densitometry. Seven of the 40 prime rs met criteria established for primer selection. However, only primer s OPE-4 (5' GTGACATGCC-3') and OPE-20 (5'-AACGGTGACC-3') allowed diffe rentiation between all 19 ATCC bacterial species included in the study . The other five primers were restricted to either gram-negative bacte ria (OPA-7, OPA-14), Staphylococcus species (OPA-13, OPA-14, OPA-ls), or Streptococcus species (OPA-3). Primers OPE-4 and OPE-20 were furthe r evaluated using 84 wild-type isolates. A bacterial species identific ation scheme was developed based on characteristic polymorphic DNA fra gments obtained with primers OPE-4 and OPE-20. Results of this study s uggest that RAPD fingerprinting has the potential for being developed into a rapid and accurate method for species identification of bacteri a in milk.