Rapid identification of Lactobacillus nantensis, Lactobacillus spicheri and Lactobacillus hammesii species using species-specific primers

Based on the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR), an identification tool for rapid differentiation of Lactobacillus nantensis, Lactobacillus spicheri and Lactobacillus hammesii, species isolated recently from French sourdough was developed. The DNA fragments containing ISRs were amplified with primers pairs 16S/p2 and 23S/p7. Clone libraries of the PCR-amplified rDNA with these primers were constructed using a pCR2.1 TA cloning kit and sequenced. The DNA sequences obtained were analyzed and species-specific primers were designed from these sequences. Two PCR amplicons, which were designated small ISR (S-ISR) and large ISR (L-ISR), were obtained for all Lactobacillus species studied. The L-ISR sequence reveale2d the presence of two tRNA genes, tRNAAla and tRNAIle. Species-specific primers designed allowed rapid identification of these species. The specificity of these primers was positively demonstrated as no response was obtained for more than 200 other species tested.