Studies on Detection of Bacteria in foods with Oligonudeotides Derived from Alanine Racemase Gene

丙氨酸消旋酶基因寡核苷酸检测食品中细菌的研究

• 批准号: 05558002
• 负责人: YOKOIGAWA Kumio
• 金额: $ 3.14万
• 依托单位: Nara Women's University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-05558002/
• 关键词: alanine racemase; food hygiene; polymerase chain reaction; bacteria; foods; detection; ハイブリダイゼーション; DNAプローブ; 生菌数測定

To detect bacteria in foods simply and convenietly, we examined the conditions for amplification of a gene fragment of alanine racemase by polymerase chain reaction. Several primers corresponding to the regions of the active site and His168 to Glu174 of alanine racemase were synthesized and used for this study. Specific amplification of the gene fragment (400bp) was achieved when the PCR was performed at 94 C for 20 s for denaturation, at 54 C for 30 s for annealing, and at 72 C for 180 s for extension. the mehod was applicable for amplification of the gene fragment from various bacteria. When foods such as milk and mineral water containing bacteria was used as templates for the PCR,one bacterial cells in the foods was detected by the PCR.This method is useful for detection of bacteria in foods.

为了简便、快速地检测食品中的细菌，我们研究了聚合酶链反应扩增丙氨酸消旋酶基因片段的条件。合成了对应于丙氨酸消旋酶活性位点和His168至Glu174区域的几种引物，并用于本研究。在94 ℃变性20 s，54 ℃退火30 s，72 ℃延伸180 s的条件下，可获得400 bp的特异性扩增片段。该方法适用于多种细菌的基因片段扩增。以牛奶、矿泉水等含菌食品为模板进行PCR检测，均能检测到其中一个细菌细胞，该方法可用于食品中细菌的检测。

项目成果

Kumio Yokoigawa: "Thermolabile alanine racemase from a psychrotroph:purification and properties." Biosci.Biotech.Bioeng.57. 93-97 (1993)

Kumio Yokoikawa：“来自耐冷菌的耐热丙氨酸消旋酶：纯化和特性。”

横井川久己男: "Thermolabile alanine racemase from a psychrotroph,Pseudomonas fluorescens:purification and properties" Biosci.Biotech.Bioeng.57. 93-97 (1993)

Hisao Yokoikawa：“来自耐冷菌荧光假单胞菌的耐热丙氨酸消旋酶：纯化和特性”Biosci.Biotech.Bioeng.57 (1993)。

林英姫: "A new amino acid racemase with threonine α-epimerase activity from Pseudomonas putida:purification and characterization" J.Bacteriology. 175. 4213-4217 (1993)

Hideki Hayashi：“来自恶臭假单胞菌的具有苏氨酸 α-差向异构酶活性的新氨基酸消旋酶：纯化和表征”J.Bacteriology。175. 4213-4217 (1993)

Kumio Yokoigawa, Hiroyasu Kawai, Kinji Endo, Young-Hee Lim, Nobuyoshi Esaki, and kenji Soda: "Thermolabile alanine racemase from a psychrotroph : purification and properties." Biosci.Biotech.Bioeng.57. 93-97 (1993)

Kumio Yokoikawa、Hiroyasu Kawai、Kinji Endo、Young-Hee Lim、Nobuyoshi Esaki 和 kenji Soda：“来自耐冷菌的耐热丙氨酸消旋酶：纯化和特性。”

Kumio Yokoigawa: "Thermolabile alanine racemase from a psychrotroph: purification and properties." Biosci. Biotech. Bioeng.57. 93-97 (1993)

Kumio Yokoikawa：“来自耐冷菌的耐热丙氨酸消旋酶：纯化和特性。”