Monitoring of Psychrotrophs in Foods by Polymerase Chain Reaction

通过聚合酶链反应监测食品中的耐冷菌

• 批准号: 06680019
• 负责人: YOKOIGAWA Kumio
• 金额: $ 1.34万
• 依托单位: Nara Women's University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06680019/
• 关键词: Alanine racemase; Psychrotroph; Food Hygiene; ポリメラーゼチェインリアクション; 細菌検査

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.

用几种简并寡核苷酸引物，通过聚合酶链反应（PCR）检测了来自各种细菌的典型原核酶丙氨酸消旋酶的基因片段的扩增。根据枯草芽孢杆菌、嗜热脂肪芽孢杆菌和鼠伤寒沙门氏菌的酶的Lys 41和His 168残基侧翼氨基酸的保守序列设计引物。发现用适当考虑这些丙氨酸消旋酶基因中的密码子使用而设计的一对引物进行PCR，从嗜冷芽孢杆菌、阴沟肠杆菌、解冷糖芽孢杆菌和大肠杆菌的基因组DNA中产生大小约为390个碱基对的特异性扩增产物。这4个扩增产物经DNA测序证实为丙氨酸消旋酶的基因片段;在4个PCR产物的翻译序列中也发现了已知丙氨酸消旋酶中存在的保守氨基酸残基。从枯草B.、嗜热脂肪B.、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，但从面包酵母和尼日尔曲霉中没有扩增出。该方法适用于牛奶中细菌的检测，在4 h内可检测到牛奶中的单个细菌细胞（解冷糖芽孢杆菌）。

项目成果

Yoko Murakami: "Induction of Freeze-sensitive Mutants from a Freeze-Tolerant Yeast,Torulaspora delbrueckii" Biosci,Biotech.Biochem.58. 206-207 (1994)

Yoko Murakami：“从耐冷冻酵母中诱导冷冻敏感突变体，德氏圆酵母”Biosci，Biotech.Biochem.58。

Yoko Okubo: "Lability of Alanine Racemase from a Psychrotoph" J.Home Econ.Jpn.46. 1135-1140 (1995)

Yoko Okubo：“来自 Psychrotoph 的丙氨酸消旋酶的不稳定性”J.Home Econ.Jpn.46。

横井川久己男: "食品の細菌学的検査におけるバイオテクノロジー" New Food Industry. 37. 32-38 (1995)

横井川久男：“食品细菌学检测中的生物技术”《新食品工业》37. 32-38 (1995)。

Kumio Yokoigawa: "Biotechnology of Bacteriological Examination in Foods" New Food Industry. 37. 32-38 (1995)

横井川久美雄：《食品中细菌检验的生物技术》新食品工业。