公开(公告)号：US06225074B1

公开(公告)日：2001-05-01

申请号：US08932072

申请日：1997-09-17

申请人： Dennis Wright ,  David J. Phelps

发明人： Dennis Wright ,  David J. Phelps

IPC分类号： C12Q148

CPC分类号： C12Q1/26 ,  G01N2333/91057 ,  G01N2333/916 ,  G01N2333/924 ,  Y10S435/81

摘要： A direct assay for chloramphenicol acetyl transferase (CAT) has been presented wherein the assay reagent comprises chloramphenicol, an acyl CoA compound, and a tetrazolium salt, and wherein the reagent does not have any added coupling redox enzymes. In one embodiment, the reagent is mixed with the test sample and the presence of CAT is detected by an optical response. In a second embodiment, the reagent is mixed with a test sample containing CAT and the optical response is quantitated by comparison with standards to measure CAT activity in the test sample. In another embodiment, an exogenous electron carrier such as phenazines may be used to enhance the detection of CAT. The assay for the presence or activity of CAT can be used in a high-throughput screening assay or to detect CAT as a reporter gene for measuring the expression of a gene of interest. Kits containing the reagents are also provided.

摘要翻译： 已经提出了用于氯霉素乙酰转移酶（CAT）的直接测定法，其中测定试剂包括氯霉素，酰基辅酶A化合物和四唑鎓盐，并且其中所述试剂不具有任何添加的偶联氧化还原酶。 在一个实施方案中，将试剂与测试样品混合，并通过光学响应检测CAT的存在。 在第二个实施方案中，将试剂与含有CAT的测试样品混合，并通过与标准品进行比较来定量光学响应以测量测试样品中的CAT活性。 在另一个实施方案中，外源电子载体如吩嗪可用于增强CAT的检测。 CAT的存在或活性的测定可用于高通量筛选测定或检测CAT作为用于测量感兴趣的基因表达的报告基因。 还提供了含有试剂的试剂盒。