Monitoring of Psychrotrophs in Foods by Polymerase Chain Reaction

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Amplification of a gene fragment of a typical prokaryotic enzyme, alanine racemase, from various bacteria was examined by polymerase chain reaction (PCR) with several degenerate oligonucleotide primers. The primers were designed based upon the conserved sequences of amino acids flanking the Lys 41 and His 168 residues of the enzymes from Bacillus subtilis, Bacillus stearothermophilus, and Salmonella typhimurium. PCR with a pair of primers designed in due consideration of the codon usage in these alanine racemase genes was found to produce a specifically amplified product with a size of approximately 390-base pair from genomic DNAs of Bacillus psychrophilus, Enterobacter cloacae, Bacillus psychrosaccharolyticus, and Escherichia coli. These four amplified products were confirmed as the gene fragments of alanine racemases by DNA sequencing ; the conserved amino acid residues present in known alanine racemases were also found in the four translated sequences of the PCR products. About 390-base pair DNA fragments were also amplified from B.subtilis, B.stearothermophilus, Serratia marcescens, and Pseudomonas fluorescens, but not from a bakers' yeast and Aspergillus niger. The PCR method was applicable to the detection of bacteria in cow's milk : single bacterial cell (Bacillus psychrosaccharolyticus) in cow's milk was detected within 4h.