DNA probe for Lactobacillus delbrueckii subsp. lactis

Lactobacilli are of great commercial value because they are widely used as starters in food fermentation. The species Lactobacillus delbrueckii, which comprises the three subspecies bulgaricus, lactis and delbrueckii, is important in dairy products and vegetables. The subspecies bulgaricus is present in yogurt, and subspecies lactis is recovered from whey, starter cultures and cheeses (Stiles & Holzapfel, 1997). It is unusual to recover Lb. delbrueckii subsp. delbrueckii (Lb. delbrueckii) from dairy products, from which Lb. delbrueckii subsp. lactis (Lb. lactis) and subsp. bulgaricus (Lb. bulgaricus) are typically isolated. However, given the similarity of these two latter subspecies, a clear identi®cation of isolates on the basis of phenotype criteria alone is often problematic (Dellaglio, 1989; Millie' re et al. 1996). The use of DNA probes and methods based on polymerase chain reaction (PCR) have greatly facilitated diagnostic identi®cation of bacteria. For the lactobacilli, DNA probes have been described for Lb. helveticus, Lb. acidophilus, Lb. fermentum, Lb. plantarum and most of the other Lactobacillus species (Pot et al. 1994; Tailliez et al. 1994; Quere et al. 1997). For Lb. delbrueckii, an EcoRI DNA fragment of the plasmid pY85 was used as a probe for this species, although it was not able to discriminate its three subspecies (Delley et al. 1990). In our laboratory, a speci®c ampli®cation of a DNA fragment of C1±7 kbp using universal primers for the ampli®cation of ribosomal RNA (rRNA) genes was found only for dairy isolates of Lb. lactis and not for Lb. bulgaricus, Lb. delbrueckii, Lb. helveticus and Lb. acidophilus (G. Giraffa, P. de Vecchi and L. Rossetti, unpublished results). This prompted us to test the possible use of this fragment as a speci®c DNA probe for Lb. lactis. To this end, Southern and dot blot hybridization experiments were carried out with total DNA of several strains belonging to different lactic acid bacteria (LAB) species.