Dimitrov D H, Graham D Y, Estes M K
J Infect Dis. 1985 Aug;152(2):293-300. doi: 10.1093/infdis/152.2.293.
We developed a dot-blot hybridization assay to detect rotaviral RNA sequences in tissue culture or in clinical samples. 32P-labeled cloned cDNA probes of the simian rotavirus SA11 specifically detected rotaviral RNA sequences and were more sensitive for detecting SA11 than was the commercial enzyme-linked immunosorbent assay Rotazyme test. A full-length probe of SA11 gene 6 detected 2.5 X 10(5) SA11 particles or approximately 0.27 ng of purified SA11 dsRNA. Combined probes from genes 6 and 9 detected 0.135 ng of purified SA11 dsRNA. The assay detected group A rotaviruses from different subgroups and serotypes, but the sensitivity of RNA detection varied from 0.5 to 31 ng when RNA from heterologous strains of virus was analyzed. An analysis of coded stool samples correctly identified 31 (91%) of 34 samples positive for rotavirus by electron microscopy and 100% of 26 samples negative for rotavirus by electron microscopy. Preliminary experiments also showed the assay has potential to directly characterize (subgroup and serotype) rotaviral isolates.
我们开发了一种斑点杂交检测法，用于检测组织培养物或临床样本中的轮状病毒RNA序列。猿猴轮状病毒SA11的32P标记克隆cDNA探针能特异性检测轮状病毒RNA序列，且在检测SA11时比商业酶联免疫吸附测定Rotazyme试验更灵敏。SA11基因6的全长探针能检测到2.5×10(5)个SA11颗粒或约0.27 ng纯化的SA11双链RNA。来自基因6和9的组合探针能检测到0.135 ng纯化的SA11双链RNA。该检测法能检测不同亚组和血清型的A组轮状病毒，但分析来自异源病毒株的RNA时，RNA检测灵敏度在0.5至31 ng之间变化。对编码粪便样本的分析正确鉴定出34份经电子显微镜检测轮状病毒呈阳性的样本中的31份（91%），以及26份经电子显微镜检测轮状病毒呈阴性的样本中的100%。初步实验还表明该检测法有直接鉴定（亚组和血清型）轮状病毒分离株的潜力。
