Herrington C S, Burns J, Graham A K, Bhatt B, McGee J O
University of Oxford, Nuffield Department of Pathology and Bacteriology, John Radcliffe Hospital.
J Clin Pathol. 1989 Jun;42(6):601-6. doi: 10.1136/jcp.42.6.601.
A method was developed for the simultaneous detection of viral and human DNA in contrasting colours in routine formalin fixed, paraffin wax embedded biopsy specimens. This was achieved by non-isotopic in situ hybridisation (NISH) with a biotinylated Y chromosome probe and digoxigenin labelled probe for human papilloma virus type 6 (HPV 6). The tissues studied were peripheral lymphocytes, tonsil, and penile warts. The hybridisation signals produced by biotinylated probes were visualised in red using streptavidin peroxidase and those produced by digoxigenin labelled probes as a blue/black colour using anti-digoxigenin alkaline phosphatase. In lymphocytes and tonsil 95-100% of cells had a detectable Y chromosome; in warts only 60-70% of infected keratinocytes near the skin surface had a demonstrable Y chromosome. This suggests that this chromosome is lost or occluded in cell maturation. In simultaneous double hybridisation with both probes, HPV and Y sequences were demonstrable within the same nucleus in penile warts. This technique permits the simultaneous differential detection of two nuclei acid sequences in interphase nuclei and will have application in analysis of putative dual HPV infections and in determining the intranuclear spatial relations between nucleic acids in interphase nuclei.
已开发出一种方法，可在常规福尔马林固定、石蜡包埋的活检标本中同时以对比颜色检测病毒DNA和人类DNA。这是通过用生物素化的Y染色体探针和地高辛配体标记的人乳头瘤病毒6型（HPV 6）探针进行非同位素原位杂交（NISH）来实现的。所研究的组织为外周淋巴细胞、扁桃体和阴茎疣。生物素化探针产生的杂交信号用链霉抗生物素蛋白过氧化物酶显示为红色，地高辛配体标记探针产生的信号用抗地高辛配体碱性磷酸酶显示为蓝/黑色。在淋巴细胞和扁桃体中，95% - 100%的细胞有可检测到的Y染色体；在疣中，仅皮肤表面附近60% - 70%受感染的角质形成细胞有可显示的Y染色体。这表明该染色体在细胞成熟过程中丢失或被封闭。在与两种探针同时进行的双重杂交中，HPV和Y序列在阴茎疣的同一细胞核内均可显示。该技术允许在间期核中同时差异检测两种核酸序列，并将应用于分析假定的双重HPV感染以及确定间期核中核酸之间的核内空间关系。
