Begaud E, Germani Y
Institut Pasteur de Nouvelle Calédonie, Laboratoire des Bactéries Entéropathogènes, Nouméa.
Res Microbiol. 1992 Mar-Apr;143(3):315-25. doi: 10.1016/0923-2508(92)90023-h.
The present study describes acetylaminofluorene(AAF)-modified DNA probes for the identification of heat-labile (LT) and heat-stable (porcine STp and human STh) toxins from enterotoxigenic Escherichia coli (ETEC). AAF probes were compared with established biotinylated probes and bioassays. Ultracentrifugation was not necessary in the preparation of AAF-labelled probes, and the procedures, i.e. chemical modification of probes, hybridization and immunodetection steps, were optimized to detect ETEC by colony hybridization or direct dot blot techniques. The method combines chemical labelling (covalent attachment of AAF group to guanosine is achieved by treatment of DNA with N-acetoxy-2-acetylaminofluorene) and detection of the hybridized target DNA by means of anti-AAF monoclonal antibodies and alkaline phosphatase-labelled second antibodies.
本研究描述了用于鉴定产肠毒素大肠杆菌（ETEC）热不稳定毒素（LT）和热稳定毒素（猪STp和人STh）的乙酰氨基芴（AAF）修饰的DNA探针。将AAF探针与已建立的生物素化探针和生物测定法进行了比较。制备AAF标记的探针无需超速离心，并且对探针的化学修饰、杂交和免疫检测步骤等程序进行了优化，以通过菌落杂交或直接斑点印迹技术检测ETEC。该方法结合了化学标记（通过用N - 乙酰氧基 - 2 - 乙酰氨基芴处理DNA，将AAF基团共价连接到鸟苷上）以及借助抗AAF单克隆抗体和碱性磷酸酶标记的二抗检测杂交的靶DNA。
