简便、敏感的地高辛标记DNA探针原位分子杂交技术

A Sensitive and Convenient Method of in Situ Hybridization by Using Digoxigenin Labelling Probe

摘要: 本文应用人乳头瘤病毒6B和11型基因探针原位分子杂交技术检测了57例尖锐湿疣和假性湿疣。同时用免疫组化ABC法做对照检测。探针标记选择地高辛随机引物延伸法。原位分子杂交的主要步骤包括:石蜡切片的预处理;杂交;免疫检测及显色。结果显示:在尖锐湿疣中,人乳头瘤病毒的阳性率为94.4％;免疫组化核壳抗原检测阳性率为47.2％;假性湿疣杂交结果均为阴性。结果表明:原位分子杂交方法比免疫组化方法敏感,提高了阳性率。本实验过程中遇到并试图解决的主要技术问题有:(1)防止脱片;(2)蛋白酶K的浓度;(3)显色时间的掌握;(4)左旋脒唑的剂量。

Abstract: HPV6B and 11 probes were used to detect 57 samples of condylomata acuminata and pseudocondylomata with in situ hybridization. Immunohistochemical staining for HPV-Ag was also studied as control group. HPV6B and 11 DNA were labelled by digoxigenin-11-dUTP with random primer extension. The major steps of in situ hybridizition consisted of pretreatment of slides, hybridization and immunological detection. The results showed positive rate of HPV6B and 11 were 94.4％ in condyloma, positive rate of HPV-Ag was only 47.2％. In the experimental process, we met and tried to solve some problems such as preventing section from falling off, concentration of proteinase K dosage of L-levamisole, and the time of coloring period.