霍乱毒素基因酶标寡核苷酸探针的研制
Development of an enzyme-labeled oligonucleotide probe for the cholera toxin gene.
作者信息
Yoh M, Miyagi K, Matsumoto Y, Hayashi K, Takarada Y, Yamamoto K, Honda T
机构信息
Research Institute for Microbial Diseases, Osaka University, Japan.
出版信息
J Clin Microbiol. 1993 May;31(5):1312-4. doi: 10.1128/jcm.31.5.1312-1314.1993.
Abstract
An alkaline phosphatase-conjugated 30-mer oligonucleotide probe was developed to detect the cholera toxin gene (ctx) in Vibrio cholerae O1. For rapid identification, V. cholerae O1 was grown on selective agar (thiosulfate-citrate-bile salts agar) or in alkaline peptone water and organisms were transferred directly to nylon membranes. Lysis of cells, denaturation of DNA, neutralization, and hybridization were carried out on the membrane. These procedures required only 3 h for completion. The results of the hybridization test with 88 clinical and 20 environmental isolates agreed almost exactly with the results of the immunological tests (anti-cholera toxin antibody-sensitized latex agglutination tests). The specificity of the probe was also tested with strains of enterotoxigenic Escherichia coli, V. cholerae non-O1, and Vibrio mimicus.
摘要
开发了一种碱性磷酸酶偶联的30聚体寡核苷酸探针，用于检测霍乱弧菌O1中的霍乱毒素基因（ctx）。为了快速鉴定，将霍乱弧菌O1在选择性琼脂（硫代硫酸盐-柠檬酸盐-胆盐琼脂）上或碱性蛋白胨水中培养，然后将菌直接转移到尼龙膜上。在膜上进行细胞裂解、DNA变性、中和及杂交。这些步骤仅需3小时即可完成。对88株临床分离株和20株环境分离株进行杂交试验的结果与免疫试验（抗霍乱毒素抗体致敏乳胶凝集试验）的结果几乎完全一致。还用产肠毒素大肠杆菌、非O1群霍乱弧菌和拟态弧菌菌株测试了该探针的特异性。
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