Lee L H, Ting L J, Shien J H, Shieh H K
Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan.
J Clin Microbiol. 1994 May;32(5):1268-72. doi: 10.1128/jcm.32.5.1268-1272.1994.
An assay protocol based on single-tube, noninterrupted reverse transcription-PCR (RT-PCR) for the detection of infectious bursal disease virus (IBDV) is described. After the conditions for RT-PCR had been optimized, a primer set framing a region within the gene coding for IBDV VP2 protein was used to amplify a 318-bp fragment of the IBDV genome. Amplified product was detected with three strains of IBDV, whereas none was obtained from uninfected bursal tissue or seven unrelated avian viruses. The sensitivity of this RT-PCR was tested with purified viral RNA from three strains of IBDV. The detection limit was 10 fg in an ethidium bromide-stained gel. In addition, this assay system was used to detect IBDV in bursal-tissue specimens from commercially reared chickens. The identity of the amplified products from the tissue specimen preparation was determined by using a rapid, simple procedure in which internally nested, end-labeled probes were used.
本文描述了一种基于单管、不间断逆转录-聚合酶链反应（RT-PCR）检测传染性法氏囊病病毒（IBDV）的检测方案。在优化RT-PCR条件后，使用一组引物扩增IBDV基因组中编码VP2蛋白基因内的一个区域，获得了一条318 bp的IBDV基因组片段。该扩增产物能被三株IBDV检测到，而未感染的法氏囊组织或七种无关禽病毒均未得到扩增产物。用三株IBDV的纯化病毒RNA检测了该RT-PCR的灵敏度。在溴化乙锭染色凝胶中的检测限为10 fg。此外，该检测系统用于检测商业饲养鸡的法氏囊组织标本中的IBDV。通过使用一种快速、简单的程序，即使用内部嵌套的、末端标记的探针，确定了组织标本制备中扩增产物的同一性。
