Miyamoto T, Miwa H, Hatano S
Department of Food Science and Technology, Faculty of Agriculture, Kyushu University, Fukuoka, Japan.
Appl Environ Microbiol. 1990 May;56(5):1480-4. doi: 10.1128/aem.56.5.1480-1484.1990.
An improved fluorogenic assay for the rapid detection of Vibrio parahaemolyticus was developed. In the improved assay, the enrichment of V. parahaemolyticus was carried out in arabinose-glucuronate medium (0.5% arabinose, 0.25% glucuronate, 0.1% polypeptone, 0.1% yeast extract, 0.1% ammonium sulfate, 2% NaCl, 2 micrograms of polymyxin B sulfate per ml, pH 8.5) at 37 degrees C. After the cultivation, the trypsinlike activity of the bacteria was measured by fluorescence with the fluorogenic substrate benzoyl-L-arginine-7-aminomethylcoumarin. Even in the presence of 3 x 10(5) cells of Vibrio alginolyticus, 20 cells of V. parahaemolyticus were clearly detected after a 6-h enrichment cultivation by the assay. Fifty contaminated samples of 14 seafoods were examined for V. parahaemolyticus by the fluorogenic assay after enrichment cultivation for 6 or 8 h. The results were then compared with those obtained by the conventional bromothymol blue Teepol agar assay and the most-probable-number method. There was a linear relationship between trypsinlike activity measured by the assay and the number of V. parahaemolyticus cells in seafood as determined by the bromothymol blue Teepol agar and most-probable-number methods. Correlation coefficients were 0.95 and 0.93 after a 6-h cultivation and an 8-h cultivation, respectively. The presence of 10 cells of V. parahaemolyticus per gram of seafood sample was detected after a 10-h total detection time by the fluorogenic assay.
开发了一种用于快速检测副溶血性弧菌的改进荧光测定法。在改进的测定法中，副溶血性弧菌在阿拉伯糖 - 葡萄糖醛酸培养基（0.5％阿拉伯糖，0.25％葡萄糖醛酸，0.1％蛋白胨，0.1％酵母提取物，0.1％硫酸铵，2％氯化钠，每毫升2微克硫酸多粘菌素B，pH 8.5）中于37℃下进行富集培养。培养后，使用荧光底物苯甲酰 - L - 精氨酸 - 7 - 氨基甲基香豆素通过荧光法测量细菌的类胰蛋白酶活性。即使存在3×10⁵ 溶藻弧菌细胞，通过该测定法在6小时富集培养后仍能清晰检测到20个副溶血性弧菌细胞。对14种海鲜的50个污染样本在富集培养6小时或8小时后通过荧光测定法检测副溶血性弧菌。然后将结果与通过传统的溴百里酚蓝吐温琼脂测定法和最大可能数法获得的结果进行比较。该测定法测量的类胰蛋白酶活性与通过溴百里酚蓝吐温琼脂和最大可能数法测定的海鲜中副溶血性弧菌细胞数量之间存在线性关系。6小时培养和8小时培养后的相关系数分别为0.95和0.93。通过荧光测定法在总检测时间为10小时后检测到每克海鲜样本中存在10个副溶血性弧菌细胞。
