Identifying Lactobacillus species using only phenotypic and genotypic (16S rDNA sequence analysis)
techniques yields inaccurate results. The objective of this study was to develop species-specific primers
based on randomly amplified polymorphic DNA (RAPD) fingerprinting to distinguish species within the
closely related Lactobacillus plantarum group. One of these primers, OPD-3, produced a species-specific
band that was found only in the tested Lactobacillus pentosus. This specific fragment was isolated from
agarose gel and ligated into a vector for DNA sequencing. A pair of primers, SpOPD3Lpen-F1/R1, that were
highly specific sequence-characterized-amplified-regions (SCARs) were designed according to the
nucleotide sequences of the specific RAPD marker. These primers were used for PCR analysis of the
template DNA of the Lactobacillus strains, and a single 542 bp species-specific band was found only in L.
pentosus. Using PCR, a novel species-specific primer pair is shown to rapidly, accurately and effectively
distinguish L. pentosus from other species in the L. plantarum group of probiotic bacteria.