Nucleic acid hybridization for detection of cell culture-amplified adenovirus
Nucleic acid hybridization for detection of cell culture-amplified adenovirus
复制标题
核酸杂交检测细胞培养扩增的腺病毒
DOI：
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发表时间：
1988
影响因子：
9.4
通讯作者：
R. Deibel
中科院分区：
文献类型：
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作者：
C. Huang;R. Deibel
构建了许多含有腺病毒基因组片段的重组质粒。通过核酸杂交技术测试了七个克隆探针以及总腺病毒 2 型 (Ad2) 和 Ad16 基因组 DNA 检测感染细胞中腺病毒的灵敏度和特异性。将腺病毒 DNA 点样到硝酸纤维素滤膜上并与 32P 标记的 DNA 探针杂交。探针、总 Ad2 基因组 DNA 和质粒 pAd2-H（含有 Ad2 DNA 的六邻体基因）均检测到 5 个基因组亚组（A 至 E）的 10 种参考血清型，具有相似的灵敏度。然而，质粒 pAd2-H 所需的制备时间比总 Ad2 DNA 少。探针 pAd2-F（含有 Ad2 的纤维基因）和 pAd16-BD（含有 Ad16 的 BamHI D 片段）仅与同源亚组（分别为 C 和 B）的参考血清型杂交。在细胞中扩增的 101 个患者分离株中，pAd2-H 检测到 100% 来自同源和异源亚组的所有分离株。 pAd2-F的检出率为100%（C亚组）和3.6%（A、B和D亚组），pAd16-BD的检出率为100%（B亚组）和9.4%（A、C和D亚组）。本研究还包括商业生物素化产品（Pathogen II）进行比较。
A number of recombinant plasmids containing genomic segments of adenovirus were constructed. Seven cloned probes, as well as total adenovirus type 2 (Ad2) and Ad16 genomic DNA, were tested by a nucleic acid hybridization technique for sensitivity and specificity in detecting adenoviruses in infected cells. Adenovirus DNA was spotted onto a nitrocellulose filter and hybridized with 32P-labeled DNA probes. The probes, total Ad2 genomic DNA, and plasmid pAd2-H (containing the hexon gene from Ad2 DNA) all detected 10 reference serotypes of five genomic subgroups (A through E) with similar sensitivities. However, plasmid pAd2-H required less preparation time than did total Ad2 DNA. Probes pAd2-F (containing the fiber gene from Ad2) and pAd16-BD (containing the BamHI D fragment from Ad16) hybridized only with reference serotypes from the homologous subgroups (C and B, respectively). Of 101 patient isolates amplified in cells, pAd2-H detected 100% of all isolates from both the homologous and the heterologous subgroups. The detection rates for pAd2-F were 100% (subgroup C) and 3.6% (subgroups A, B, and D), and those for pAd16-BD were 100% (subgroup B) and 9.4% (subgroups A, C, and D). A commercial biotinylated product (Pathogene II) was also included in this study for comparison.