Unger R E, Chuang R Y, Chuang L F, Doi R H, Osburn B I
Department of Medical Pharmacology, University of California, Davis 95616.
J Virol Methods. 1988 Dec;22(2-3):273-82. doi: 10.1016/0166-0934(88)90109-7.
Dot-blot and Northern blot hybridization methods to determine the genetic relatedness of United States bluetongue virus serotypes 2, 10, 11, 13, and 17 were compared. Both plasmid and insert DNA probes from cloned BTV-17 dsRNA segments 2, 5, 6, and 8 were hybridized to dsRNA from the BTV serotypes and epizootic hemorrhagic disease virus (EHDV). Stringencies of hybridization were kept identical, and experiments differed only in the method in which dsRNA was applied to the membranes (dot-blot or Northern blot). The Northern blot hybridization method yielded more consistent results, and it was visually and unequivocally shown to which dsRNA segment a cDNA probe bound, since the dsRNA segments were separated by PAGE prior to blotting and hybridization. In contrast, the dot-blot hybridization method gave less consistent results. Identical results for plasmid and insert probes were obtained for Northern blot hybridizations but not for dot-blot hybridizations. At least two probes could be used simultaneously in Northern blot hybridizations.
比较了斑点印迹法和Northern印迹杂交法，以确定美国蓝舌病毒2型、10型、11型、13型和17型的遗传相关性。来自克隆的BTV - 17双链RNA片段2、5、6和8的质粒和插入DNA探针与BTV各血清型及流行性出血病病毒（EHDV）的双链RNA进行杂交。杂交的严谨性保持一致，实验仅在将双链RNA应用于膜的方法（斑点印迹法或Northern印迹法）上有所不同。Northern印迹杂交法产生的结果更一致，并且能直观明确地显示cDNA探针与哪个双链RNA片段结合，因为双链RNA片段在印迹和杂交之前通过聚丙烯酰胺凝胶电泳（PAGE）进行了分离。相比之下，斑点印迹杂交法得到的结果不太一致。对于Northern印迹杂交，质粒和插入探针获得了相同的结果，但斑点印迹杂交则不然。在Northern印迹杂交中可以同时使用至少两种探针。
