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

Abstract

In order to demonstrate that creation of silent mutations allows a precise strain-specific detection with polymerase chain reaction, silent mutations positions were introduced in adjacent codons of the Lactobacillus helveticus CNRZ32 chromosomal pepX gene without altering the amino acid sequence of the gene product, X-prolyl dipeptidyl aminopeptidase (PepX). The mutated pepX gene was constructed with polymerase chain reaction and cloned with the thermosensitive pSA3 plasmid in L. helveticus CNRZ32 and integrated in the chromosomal DNA by homologous recombination at a restrictive temperature. Gene replacement of the wild-type pepX gene with that of the mutated variant was obtained in the second homologous recombination event. The PepX activity of mutant strains carrying the pepX gene with silent mutations did not differ from that of the wild-type strain. PCR was performed with strain-specific primers from milk and faecal samples spiked with the wild-type or mutant L. helveticus strain. For this purpose, a method for removing PCR inhibitors from faecal samples by washing steps and cell mill disruption of the bacterial cells combined with Sepharose CL-6B spin column chromatography was also developed.