Clavijo A, Thorsen J
Department of Veterinary, Microbiology and Immunology, University of Guelph, Ontario, Canada.
Vet Microbiol. 1995 Mar;43(4):295-305. doi: 10.1016/0378-1135(94)00104-5.
A 814-bp digoxigenin-labelled single stranded DNA probe was produced and utilized in slot-blot hybridization for detection of caprine arthritis encephalitis virus (CAEV) in goat synovial membrane (GSM) cell culture infected with CAEV. The sensitivity of a PCR-generated probe was compared with a random primer labelled probe. The probe with digoxigenin-dUTP incorporated in the PCR reaction mixture was more sensitive for RNA detection than the random primer probe and it was much simpler to use. The probe was applied for detection of CAEV by blot blot hybridization in peripheral blood mononuclear cells (PBMC) and macrophage cultures obtained from naturally infected goats. This technique was not sufficiently sensitive to detect the viral nucleic acid directly from PBMC or cultured macrophages. When macrophages were cultured in vitro and then cocultured with susceptible GSM cells, samples gave a positive signal in the slot-blot hybridization technique. The use of slot-blot RNA hybridization permits more convenient and rapid confirmation of CAEV isolation in susceptible cells than the conventional identification by syncytia formation.
制备了一个814 bp地高辛标记的单链DNA探针，并将其用于斑点杂交，以检测感染山羊关节炎脑炎病毒（CAEV）的山羊滑膜（GSM）细胞培养物中的CAEV。将PCR产生的探针的灵敏度与随机引物标记的探针进行了比较。在PCR反应混合物中掺入地高辛-dUTP的探针在RNA检测方面比随机引物探针更灵敏，并且使用起来要简单得多。该探针通过斑点杂交用于检测从自然感染山羊获得的外周血单核细胞（PBMC）和巨噬细胞培养物中的CAEV。该技术直接从PBMC或培养的巨噬细胞中检测病毒核酸的灵敏度不够高。当巨噬细胞在体外培养然后与易感GSM细胞共培养时，样品在斑点杂交技术中给出阳性信号。与通过形成多核巨细胞进行的传统鉴定相比，使用斑点RNA杂交可以更方便、快速地确认易感细胞中CAEV的分离。
