Gibellini D, Zerbini M, Musiani M, Venturoli S, Gentilomi G, La Placa M
Institute of Microbiology, University of Bologna, Italy.
Mol Cell Probes. 1993 Dec;7(6):453-8. doi: 10.1006/mcpr.1993.1067.
A capture hybridization technique in microplate has been developed for the identification of polymerase chain reaction (PCR) amplified B19 DNA fragment in clinical specimens. The amplified 104 bp B19 DNA fragment, located in the gene coding for structural proteins, was directly labelled during the amplification reaction by incorporation of digoxigenin-labelled dUTP. The amplified product was then captured by a probe immobilized on microplate wells. The capture hybridization reaction was visualized as an enzyme-linked immunosorbent assay using anti-digoxigenin Fab fragment labelled with peroxidase. Thirty-five serum samples were tested by our capture hybridization assay and the results were in accordance with the results obtained by Southern blot analysis of PCR amplified product. Our microplate capture hybridization assay showed a high sensitivity and reproducibility and appears to be a practical and reliable test for routine screening of B19 parvovirus DNA in clinical specimens.
已开发出一种微孔板捕获杂交技术，用于鉴定临床标本中聚合酶链反应（PCR）扩增的B19 DNA片段。位于结构蛋白编码基因中的104 bp B19 DNA扩增片段，在扩增反应过程中通过掺入地高辛标记的dUTP进行直接标记。然后，扩增产物被固定在微孔板孔上的探针捕获。捕获杂交反应通过使用过氧化物酶标记的抗地高辛Fab片段进行酶联免疫吸附测定来可视化。用我们的捕获杂交试验检测了35份血清样本，结果与PCR扩增产物的Southern印迹分析结果一致。我们的微孔板捕获杂交试验显示出高灵敏度和可重复性，似乎是临床标本中B19细小病毒DNA常规筛查的一种实用且可靠的检测方法。
