Burns J, Graham A K, Frank C, Fleming K A, Evans M F, McGee J O
University of Oxford, Nuffield Department of Pathology, John Radcliffe Hospital.
J Clin Pathol. 1987 Aug;40(8):858-64. doi: 10.1136/jcp.40.8.858.
In analysing human papilloma virus (HPV) infection of the cervix in formalin fixed paraffin sections by non-isotopic in situ hybridisation two main problems were found: detachment of sections from the glass during hybridisation and probe detection; inadequate sensitivity and inability to assess sensitivity of the in situ procedure. The first problem was investigated by assessing the efficiency of various tissue adhesives individually and in combination. The second problem was addressed by optimising conditions for DNA unmasking, hybridisation, and biotinylated probe detection. Sensitivity of the final in situ procedure developed was assessed by using the detection of pHY2.1 repeats as a built-in control. Extrapolation of data showed that less than 10 copies of HPV DNA can be visualised by these procedures. HPV nucleic acid, mainly in the form of DNA, was detected not only in koilocytic nuclei but also in suprabasal cells in condylomas and CIN lesions. HPV mRNA was also visualised in the cytoplasm (and probably also nuclei) of the same cell types. These non-isotopic in situ procedures give results comparable to those obtained with radiolabelled probes, but they are less time consuming and provide better morphological resolution.
在通过非同位素原位杂交分析福尔马林固定石蜡切片中的子宫颈人乳头瘤病毒（HPV）感染时，发现了两个主要问题：杂交和探针检测过程中切片从玻片上脱落；原位检测方法灵敏度不足且无法评估其灵敏度。通过单独或组合评估各种组织黏合剂的效果来研究第一个问题。通过优化DNA解蔽、杂交和生物素化探针检测的条件来解决第二个问题。通过使用检测pHY2.1重复序列作为内对照来评估所开发的最终原位检测方法的灵敏度。数据外推表明，通过这些方法可以检测到少于10个HPV DNA拷贝。HPV核酸主要以DNA形式存在，不仅在挖空细胞核中被检测到，在尖锐湿疣和CIN病变的基底上层细胞中也能检测到。HPV mRNA也在相同细胞类型的细胞质（可能还有细胞核）中被观察到。这些非同位素原位检测方法的结果与放射性标记探针获得的结果相当，但耗时更短且提供了更好的形态分辨率。
