Hohenadl C, Klingel K, Mertsching J, Hofschneider P H, Kandolf R
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Mol Cell Probes. 1991 Feb;5(1):11-20. doi: 10.1016/0890-8508(91)90033-g.
In this report we describe the development and application of single-stranded RNA probes for strand-specific detection of enterovirus RNA in infected heart tissue by in situ hybridization. For synthesis of RNA probes a full-length reverse-transcribed, recombinant CVB3 cDNA was inserted into the transcription vector pSPT18. Run-off transcripts of plus-strand and minus-strand orientation were produced using either T7 or SP6 RNA polymerase. Binding specificity and sensitivity of the radioactively labelled RNA probes were determined by slot-blot hybridization. Due to the high degree of genetic identity among enteroviruses, the in vitro transcribed CVB3 RNA probes hybridized with various enterovirus serotypes, including group A and B coxsackieviruses and echoviruses, which are commonly implicated in human viral heart disease. Strand-specific in situ hybridization led to detection of viral plus-strand or minus-strand RNA in infected cell cultures and in myocardial tissue sections of infected mice. In consecutive sections either viral genomic plus-strand RNA or complementary minus-strand RNA were localized in the same infected myocardial cells. In situ hybridization with enterovirus-specific and highly sensitive single-stranded RNA probes is of particular interest for the diagnosis of myocardial infections and for studies concerning viral RNA replication.
在本报告中，我们描述了单链RNA探针的开发及应用，该探针可通过原位杂交对感染心脏组织中的肠道病毒RNA进行链特异性检测。为了合成RNA探针，将全长逆转录的重组柯萨奇病毒B3（CVB3）cDNA插入转录载体pSPT18中。使用T7或SP6 RNA聚合酶产生正链和负链方向的连续转录物。通过狭缝印迹杂交确定放射性标记的RNA探针的结合特异性和敏感性。由于肠道病毒之间的高度遗传同一性，体外转录的CVB3 RNA探针可与多种肠道病毒血清型杂交，包括通常与人类病毒性心脏病有关的A组和B组柯萨奇病毒及埃可病毒。链特异性原位杂交可检测感染细胞培养物和感染小鼠心肌组织切片中的病毒正链或负链RNA。在连续切片中，病毒基因组正链RNA或互补负链RNA定位于相同的感染心肌细胞中。用肠道病毒特异性且高度敏感的单链RNA探针进行原位杂交对于心肌感染的诊断以及病毒RNA复制的研究尤为重要。
