Whitby J E, Heaton P R, Whitby H E, O'Sullivan E, Johnstone P
Rabies Research and Diagnostic Group, Department of Virology, Veterinary Laboratories Agency (Weybridge), Addlestone, Surrey, UK.
J Virol Methods. 1997 Dec;69(1-2):63-72. doi: 10.1016/s0166-0934(97)00143-2.
A rapid detection method for the six established genotypes of rabies and rabies-related viral RNA using RT-PCR-ELISA is described. The detection of digoxigenin-labelled amplified products is performed by solution hybridization to two specific, biotin-labelled, capture probes, which are complementary to the inner region of the amplification products. The capture probe and amplified product hybrid are then immobilised on a streptavidin-coated microtitre plate, bound products are detected by an anti-DIG Fab fragment conjugated to peroxidase, and colorimetric reaction automatically measured. This method was up to 100-fold more sensitive than Southern blot hybridization, detecting 0.00002 TCID50/ml of a genotype 1, classical rabies virus strain. The complete detection methodology from RT-PCR to PCR-ELISA detection could be completed within 10 h. Using this procedure, we were 100% successful in detecting 60 isolates from a representative selection of the six established genotypes from all over the world. This test is a useful additional tool for the detection of the rabies and rabies-related viruses, which is easy to perform, rapid and highly sensitive.
描述了一种使用RT-PCR-ELISA快速检测狂犬病六种既定基因型和狂犬病相关病毒RNA的方法。地高辛标记的扩增产物通过与两种特异性生物素标记的捕获探针进行溶液杂交来检测，这两种探针与扩增产物的内部区域互补。然后将捕获探针和扩增产物杂交体固定在链霉亲和素包被的微量滴定板上，通过与过氧化物酶偶联的抗地高辛Fab片段检测结合产物，并自动测量比色反应。该方法比Southern印迹杂交灵敏高达100倍，可检测到1型经典狂犬病病毒株0.00002 TCID50/ml。从RT-PCR到PCR-ELISA检测的完整检测方法可在10小时内完成。使用该程序，我们成功地100%检测了来自世界各地六种既定基因型代表性选择的60个分离株。该试验是检测狂犬病和狂犬病相关病毒的一种有用的附加工具，操作简便、快速且高度灵敏。
