Monitoring of Psychrotrophs in Foods by Polymerase Chain Reaction

用几个简并的寡核苷酸引物对不同细菌的典型原核酶丙氨酸外消旋酶基因片段的扩增进行了研究。根据枯草芽孢杆菌、嗜热脂肪芽孢杆菌和鼠伤寒沙门氏菌酶的Lys 41和His 168残基两侧的氨基酸保守序列设计了引物。根据丙氨酸消旋酶基因中密码子的用法设计的一对引物，从嗜冷芽孢杆菌、阴沟肠杆菌、嗜冷性芽孢杆菌和大肠杆菌的基因组DNA中扩增出一条大小约为390个碱基对的特异扩增产物。经DNA测序证实这4个扩增产物为丙氨酸消旋酶的基因片段，在4个翻译序列中也发现了已知丙氨酸消旋酶的保守氨基酸残基。从枯草杆菌、嗜热脂肪杆菌、粘质沙雷氏菌和荧光假单胞菌中也扩增出约390个碱基对的DNA片段，但不能从烘焙酵母和黑曲霉中扩增出。该方法适用于牛乳中细菌的检测：在4h内即可检出单个细菌细胞(芽孢杆菌)。

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.