Maxwell I H, Maxwell F
Division of Medical Oncology, University of Colorado Health Sciences Center, Denver 80262.
Biotechniques. 1994 May;16(5):876-81.
We describe a convenient infected cell hybridization assay for determining the infectious titer of a recombinant, replication-defective parvovirus. We previously generated recombinant derivatives of the autonomous parvovirus LuIII, transducing the luciferase reporter gene. Since luciferase expression is not readily detected at the single cell level, and since the recombinants cannot form plaques, we developed an alternative assay for infectious particles. Co-infection of NB324K cells with wild-type LuIII (multiplicity of infection ca. 5) and the recombinant virions allows amplification of the transducing DNA, which can be detected by hybridization with a probe for the reporter gene. Cell lysis and DNA transfer to a nylon membrane is performed in situ, in the culture dish, and hybridization is performed with a digoxigenin-labeled probe, using immunological detection. During this work we developed a conveniently modified plaque assay for wild-type LuIII that should be applicable to other lytic viruses. The modification employs a reduced volume of agarose overlay that is in turn overlaid with liquid medium, thus avoiding the need to maintain stocks of culture medium at higher than normal concentration.
我们描述了一种简便的感染细胞杂交测定法，用于确定重组的、复制缺陷型细小病毒的感染滴度。我们之前构建了自主细小病毒LuIII的重组衍生物，其中转导了荧光素酶报告基因。由于在单细胞水平上不易检测到荧光素酶的表达，且重组体不能形成噬斑，因此我们开发了一种针对感染性颗粒的替代测定法。用野生型LuIII（感染复数约为5）和重组病毒粒子共同感染NB324K细胞，可实现转导DNA的扩增，这可以通过与报告基因探针杂交来检测。细胞裂解和DNA转移至尼龙膜的操作在培养皿中原位进行，杂交则使用地高辛标记的探针，并采用免疫检测法。在这项工作中，我们开发了一种针对野生型LuIII的简便改良噬斑测定法，该方法应适用于其他裂解性病毒。这种改良方法采用了体积减小的琼脂糖覆盖层，该覆盖层再覆盖有液体培养基，从而避免了以高于正常浓度保存培养基储备的需求。
