Development of a PCR-RFLP assay for the identification of Lactococcus lactis ssp. lactis and cremoris

The development of new starter culture of Lactococcus lactis for the manufacture of fermented dairy products with unique characteristics usually requires the isolation and identification of L. lactis up to subspecies level. Therefore, a rapid and specific PCR-RFLP assay has been developed. Forward and reverse primer sets were designed targeting the conserved house keeping gene htrA and yueF encoding a trypsin-like serine protease and a non-proteolytic protein from peptidase family M16, respectively, of L. lactis . Amplicons of 265 bp and 447 bp of htrA and yueF , respectively, were subjected to restriction fragment length polymorphism analysis. Restriction of the 265 bp amplicons with Taq I produced DNA bands of 90 bp and 175 bp with ssp. lactis , and 66 bp and 199 bp with ssp. cremoris . Similarly, restriction of PCR product of 447 bp size with Alu I produced digested fragments of 125 bp and 322 bp with ssp. lactis , and 71 bp and 376 bp with ssp. cremoris . The designed primer sets were observed to be specific to L. lactis because other bacteria could not be amplified. The ssp. lactis and cremoris of L. lactis could be identified by restriction of PCR products of htrA and yueF with Taq I and Alu I, respectively.