Multiplex PCR for the detection of Lactobacillus pontis and two related species in a sourdough fermentation

A specific multiplex PCR assay based on the amplification of parts of the 1 6S rRNA molecule was designed. Primers derived from variable regions of the 16S rRNA provided a means of easily differentiating the species Lactobacil lus pontis and Lactobacillus panis, They could be clearly discriminated fro m the phylogenetically related species Lactobacillus vaginalis, Lactobacill us oris, and Lactobacillus reuteri and from other lactobacilli commonly kno wn to be present in sourdough, Other strains isolated together with L, pont is from an industrial sourdough fermentation could be clearly separated fro m these species by comparative sequence analysis and construction of a spec ific PCR primer. For a fast identification a DNA isolation protocol based o n the ultrasonic lysis of cells from single colonies was developed. To demo nstrate the potential of such techniques for tracking these organisms in a laboratory-scale fermentation, we combined the specific PCR assay with dire ct DNA extraction from the organisms in the sourdough without previous cult ivation.