Musiani M, Zerbini M, Venturoli S, Gentilomi G, Gallinella G, Manaresi E, La Placa M, D'Antuono A, Roda A, Pasini P
Institute of Microbiology, University of Bologna, Italy.
J Histochem Cytochem. 1997 May;45(5):729-35. doi: 10.1177/002215549704500511.
We developed a sensitive chemiluminescence in situ hybridization assay for detection of human papillomavirus (HPV) DNA for objective and semiquantitative evaluation of the results. The hybridization reaction was performed using either digoxigenin-, biotin-, or fluorescein-labeled probes, visualized with alkaline phosphatase as the revealing enzyme and a highly sensitive 1,2 dioxetane phosphate as chemiluminescent substrate. The light emitted from the hybridized probes was detected, analyzed, and measured using a high-performance, low light-level imaging luminograph connected to an optical microscope and to a personal computer for quantification of the photon fluxes and for image analysis. The system operated in consecutive steps: First, hybridized specimens were recorded in transmitted light. Then the net luminescent signal was recorded, and then an overlay of the two images provided by the transmitted light and by the luminescent signal allowed the spatial distribution of the target DNA to be localized, measured, and evaluated. Biopsy specimens from different pathological conditions associated with HPV, which had previously been proved positive for HPV DNA with the polymerase chain reaction (PCR), were analysed. The chemiluminescence in situ hybridization proved sensitive and specific with digoxigenin-, biotin-, or fluorescein-labeled probes, and provided an objective evaluation of the results. The results obtained with chemiluminescence in situ hybridization were also compared with results obtained with in situ hybridization with colorimetric detection, with good concordance of the data. Chemiluminescence in situ hybridization therefore offers the possibility of detecting HPV DNA with great sensitivity in biopsy specimens. Moreover, the images of the samples, stored in the computer, are a permanent record of the reaction and can also be sent for evaluation or comparison to other laboratories using computer networks.
我们开发了一种用于检测人乳头瘤病毒（HPV）DNA的灵敏化学发光原位杂交检测方法，以便对结果进行客观和半定量评估。杂交反应使用地高辛、生物素或荧光素标记的探针进行，用碱性磷酸酶作为显色酶和高灵敏度的1,2 - 二氧杂环丁烷磷酸酯作为化学发光底物进行可视化。使用连接到光学显微镜和个人计算机的高性能、低光水平成像发光仪检测、分析和测量杂交探针发出的光，以量化光子通量并进行图像分析。该系统按连续步骤操作：首先，在透射光下记录杂交标本。然后记录净发光信号，然后由透射光和发光信号提供的两个图像叠加，可使目标DNA的空间分布得以定位、测量和评估。对来自与HPV相关的不同病理状况的活检标本进行了分析，这些标本先前已通过聚合酶链反应（PCR）证明HPV DNA呈阳性。化学发光原位杂交使用地高辛、生物素或荧光素标记的探针证明是灵敏且特异的，并对结果提供了客观评估。还将化学发光原位杂交获得的结果与比色检测原位杂交获得的结果进行了比较，数据具有良好的一致性。因此，化学发光原位杂交提供了在活检标本中高灵敏度检测HPV DNA的可能性。此外，存储在计算机中的样本图像是反应的永久记录，也可以通过计算机网络发送给其他实验室进行评估或比较。
