Application of Randomly Amplified Polymorphic DNA Fingerprinting for Species Identification of Bacteria Isolated from Bovine Milk

Abstract

A polymerase chain reaction-based DNA fingerprinting system for species identification of bacteria in milk was developed using randomly amplified polymorphic DNA. A total of 108 organisms including 24 ATCC reference strains and 84 wild-type isolates belonging to gram-negative, Staphylococcus, Enterococcus, and Streptococcus species were used to develop the system. Organisms included in the study were those that are isolated frequently from milk. Forty primers from two commercially available primer kits were evaluated to determine the “ideal” primer that could be used for several bacterial species. Over 960 DNA fingerprint patterns were analyzed by laser densitometry. Seven of the 40 primers met criteria established for primer selection. However, only primers OPE-4 (5′ GTGACATGCC-3′) and OPE-20 (5′-AACGGTGACC-3′) allowed differentiation between all 19 ATCC bacterial species included in the study. The other five primers were restricted to either gram-negative bacteria (OPA-7, OPA-14), Staphylococcus species (OPA-13, OPA-14, OPA-18), or Streptococcus species (OPA-3). Primers OPE-4 and OPE-20 were further evaluated using 84 wild-type isolates. A bacterial species identification scheme was developed based on characteristic polymorphic DNA fragments obtained with primers OPE-4 and OPE-20. Results of this study suggest that RAPD fingerprinting has the potential for being developed into a rapid and accurate method for species identification of bacteria in milk.