Polymerase Chain Reaction-Based DNA Fingerprinting for Identification of Streptococcus and Enterococcus Species Isolated from Bovine Milk

Abstract

A total of 353 isolates including 304 isolates from bovine milk belonging to 8 Streptococcus and Enterococcus species, 14 American Type Culture Collection reference strains, 10 isolates from bovine milk that had atypical biochemical profiles, and 25 bovine milk isolates from Vermont were examined by polymerase chain reaction-based DNA fingerprinting. A single short oligonucleotide primer of eight bases (5′-GTAACGCC-3′) was used to produce a characteristic spectrum of amplified DNA fragments. Amplified DNA fragments were visualized following agarose gel electrophoresis and analyzed by scanning laser densitometry. Amplified DNA fragments were categorized as either primary, secondary, or variable fragments. DNA fragments observed in all isolates within a species were designated as primary [optical density > 0.3 absorbance units (AU)] and secondary fragments (optical density > 0.15 AU), while infrequent DNA fragments were designated as variable fragments (optical density >0.15 AU). Examination of fingerprint patterns revealed that each of the species evaluated possessed a unique fingerprint profile. A simple identification scheme was developed based on the occurrence of primary and secondary DNA fragments for a species. The polymerase chain reaction-based DNA fingerprinting assay was simple to perform, reliable, and reproducible. Results of this study indicate that DNA fingerprinting using polymerase chain reaction has the potential to be developed as a routine method for species identification of bacteria.