Two methods for the genetic differentiation of Lactococcus lactis ssp. lactis and cremoris based on differences in the 16S rRNA gene sequence

Abstract The 16S ribosomal RNA gene sequences of Lactococcus lactis ssp. lactis and ssp. cremoris differ by 9–10 bp (depending on strain), within the first 200 bp of the sequence. These differences were used to develop two methods of genetically differentiating lactis and cremoris strains. Primers to conserved sequences in the 16S rRNA gene were used in a PCR reaction to amplify fragments of the 16S rRNA gene. A single base difference at position 180 of the sequence was utilised to develop a ligase chain reaction to differentiate lactis and cremoris sequences. The second method involved digestion of the amplified fragments with restriction endonucleases specific for either the lactis or cremoris sequence. Resolution of the digested fragments on an agarose gel allowed the strains to be identified as genetically lactis or cremoris. This method was used to examine lactococci isolated from raw milk. Of 31 raw milk strains examined, 21 contained the cremoris 16S rRNA sequence, however, all 31 strains exhibited the phenotypic characteristics of the lactis subspecies.