Johnson M E, Paul P S, Gorziglia M, Rosenbusch R
Veterinary Medical Research Institute, Iowa State University, Ames 50011.
Vet Microbiol. 1990 Sep;24(3-4):307-26. doi: 10.1016/0378-1135(90)90180-4.
A dot blot hybridization assay is described for the detection and differentiation of porcine rotavirus serotypes. Recombinant complementary DNA (cDNA) representing gene 9 (the gene encoding the neutralization antigens in VP7 glycoprotein) from OSU (porcine rotavirus serotype 1) and Gottfried (porcine rotavirus serotype 2) strains were used to determine the optimal hybridization conditions which allow specific detection of group A porcine rotaviruses. Probes were prepared by excision of the inserts from the recombinant plasmids and radiolabeling of cDNA with 32P by the random primer extension method. Probes were hybridized at various stringencies with viral RNA from different rotavirus serotypes bound to nylon membranes. Hybridization at low stringency (26% base pair mismatch for stable hybrid formation) had high sensitivity but low specificity. Hybridization at high stringency (16% base pair mismatch for stable hybrid formation) produced high specificity but decreased the sensitivity observed at low stringency. Probes were specific for rotavirus at both stringencies and did not hybridize with nucleic acids from other porcine viruses. Subgenomic gene 9 fragments were then tested to provide more specific probes. A 322 bp fragment from OSU gene 9 between nucleotides 382 and 704 and a 266 bp fragment from Gottfried gene 9 between nucleotides 230 and 496 were found to be specific as hybridization probes. These studies demonstrated the feasibility of the dot blot hybridization assay using subgenomic fragments of gene 9 to detect and differentiate serotypes of porcine rotavirus. Additional studies are warranted to further evaluate the sensitivity and the capability of these probes to detect porcine field isolates of the same serotype.
本文描述了一种用于检测和区分猪轮状病毒血清型的斑点杂交试验。使用代表来自OSU（猪轮状病毒血清型1）和Gottfried（猪轮状病毒血清型2）毒株的基因9（编码VP7糖蛋白中和抗原的基因）的重组互补DNA（cDNA）来确定最佳杂交条件，以实现对A组猪轮状病毒的特异性检测。通过从重组质粒中切除插入片段并用随机引物延伸法用32P对cDNA进行放射性标记来制备探针。探针在不同的严谨性条件下与结合在尼龙膜上的来自不同轮状病毒血清型的病毒RNA进行杂交。低严谨性杂交（形成稳定杂交体的碱基对错配率为26%）具有高灵敏度但低特异性。高严谨性杂交（形成稳定杂交体的碱基对错配率为16%）产生高特异性，但降低了在低严谨性下观察到的灵敏度。探针在两种严谨性条件下对轮状病毒均具有特异性，且不与来自其他猪病毒的核酸杂交。然后测试亚基因组基因9片段以提供更特异的探针。发现来自OSU基因9在核苷酸382和704之间的322 bp片段以及来自Gottfried基因9在核苷酸230和496之间的266 bp片段作为杂交探针具有特异性。这些研究证明了使用基因9的亚基因组片段进行斑点杂交试验来检测和区分猪轮状病毒血清型的可行性。有必要进行进一步研究以进一步评估这些探针检测相同血清型猪田间分离株的灵敏度和能力。
