Murakami T, Hagiwara T, Yamamoto K, Hattori J, Kasami M, Utsumi M, Kaneda T
Department of Clinical Research, Nagoya National Hospital (Tokai Area Central Hospital for AIDS Treatment and Research), 4-1-1 Sannomaru, Naka-ku, Nagoya, 460-0001, Aichi, Japan.
J Pathol. 2001 May;194(1):130-5. doi: 10.1002/path.843.
A novel in situ hybridization (ISH) method for detecting human immunodeficiency virus-1 (HIV-1) was developed by applying a peptide nucleic acid (PNA) probe and a catalysed signal amplification (CSA) method. The PNA probe used in the present study possessed 15 base sequences of the HIV-1 protease gene, and the 5' end of the probe was labelled with the fluorescein isothiocyanate (FITC) molecule. The hybridized probe was detected by sequential reactions of the following antibodies and reagents: horseradish peroxidase (HRP)-conjugated anti-FITC antibody, biotinylated tyramide (first amplification), HRP-labelled streptavidin, biotinylated tyramide (second amplification), and streptavidin-conjugated Alexa 488. The signal of Alexa 488 was finally detected by fluorescence microscopy. HIV-1-related dotted signals were clearly obtained in HIV-1 persistently infected cell lines, MOLT4-III(B) and ACH-2, and CD4-positive T lymphocytes from AIDS patients. For light microscopy, HRP-labelled streptavidin was reacted instead of streptavidin-conjugated Alexa 488 at the final treatment, followed by diaminobenzidine as chromogen. This method can detect HIV-1 in either blood smear samples or paraffin-embedded autopsy tissue and is useful as a sensitive non-radioactive method for in situ hybridization.
通过应用肽核酸（PNA）探针和催化信号放大（CSA）方法，开发了一种用于检测人类免疫缺陷病毒1型（HIV-1）的新型原位杂交（ISH）方法。本研究中使用的PNA探针具有HIV-1蛋白酶基因的15个碱基序列，并且探针的5'端用异硫氰酸荧光素（FITC）分子标记。通过以下抗体和试剂的顺序反应检测杂交探针：辣根过氧化物酶（HRP）偶联的抗FITC抗体、生物素化酪胺（第一次放大）、HRP标记的链霉亲和素、生物素化酪胺（第二次放大）以及链霉亲和素偶联的Alexa 488。最终通过荧光显微镜检测Alexa 488的信号。在HIV-1持续感染的细胞系MOLT4-III(B)和ACH-2以及艾滋病患者的CD4阳性T淋巴细胞中清晰地获得了HIV-1相关的点状信号。对于光学显微镜检查，在最终处理时用HRP标记的链霉亲和素代替链霉亲和素偶联的Alexa 488进行反应，随后用二氨基联苯胺作为显色剂。该方法可检测血涂片样本或石蜡包埋的尸检组织中的HIV-1，作为一种敏感的非放射性原位杂交方法很有用。
