Genetic labeling of lactobacilli in a food grade manner for strain-specific detection of industrial starters and probiotic strains

in order to demonstrate that creation of silent mutations allows a precise strain-specific detection with polymerase chain reaction silent mutations p ositions were introduced in adjacent codons of the Lactobacillus helveticus CNRZ32 chromosomal pepX gene without altering She amino acid sequence of t he gene product, X-prolyl dipeptidyl aminopeptidase (PepX). The mutated pep X gene was constructed with polymerase chain reaction and cloned with the t hermosensitive pSA3 plasmid in L. helveticus CNRZ32 and integrated in the c hromosomal DNA by homologous recombination at a restrictive temperature. Ge ne replacement of the wild-type pepX gene with that of the mutated variant was obtained in the second homologous recombination event. The PepX activit y of mutant strains carrying the pepX gene with silent mutations did not di ffer from that of the wild-type strain. PCR was performed with strain-speci fic primers from milk and faecal samples spiked with the wild-type or mutan t L, helveticus strain. For this purpose a method for removing PCR inhibito rs from faecal samples by washing steps and cell mill disruption of the bac terial cells combined with Sepharose CL-6B spin column chromatography was a lso developed. (C) 2001 Academic.