Unger E R, Chandler F W, Chenggis M L, Ou C Y, Warfield D T, Feorino P M
Department of Pathology and Laboratory Medicine, Emory University, Atlanta, Georgia.
Mod Pathol. 1989 May;2(3):200-4.
A colorimetric method of in situ hybridization has been developed for the rapid detection of human immunodeficiency virus (HIV) in formalin-fixed paraffin-embedded material. Following optimization of digestion conditions, biotin-labeled DNA probes are detected with an alkaline phosphatase conjugate. The method is verified using fixed paraffin-embedded cell blocks of HIV-infected and uninfected lymphocyte cell cultures. Hybridization specifically detects both viral RNA and proviral DNA. Formalin fixation for intervals up to 21 d did not significantly hamper the signal under the appropriate digestion conditions; however, Trump's fixation for even 12 h greatly reduced the intensity of the hybridization. This technique for in situ hybridization is amenable to automation, provides results within 6 h, and results in good morphologic preservation. A key feature of the technique is the use of human placental DNA as an endogenous positive control to optimize the empirically determined conditions for protein digestion.
已开发出一种比色原位杂交方法，用于在福尔马林固定石蜡包埋材料中快速检测人类免疫缺陷病毒（HIV）。在优化消化条件后，用碱性磷酸酶偶联物检测生物素标记的DNA探针。使用HIV感染和未感染淋巴细胞培养物的固定石蜡包埋细胞块对该方法进行验证。杂交可特异性检测病毒RNA和前病毒DNA。在适当的消化条件下，长达21天的福尔马林固定不会显著影响信号；然而，即使12小时的特朗普固定也会大大降低杂交强度。这种原位杂交技术适合自动化，6小时内即可得出结果，且形态保存良好。该技术的一个关键特征是使用人胎盘DNA作为内源性阳性对照，以优化根据经验确定的蛋白质消化条件。
