A novel method for detecting HIV-1 by non-radioactive in situ hybridization: application of a peptide nucleic acid probe and catalysed signal amplification.
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.
Murakami
T
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.
Hagiwara
T
T
Yamamoto
K
K
Hattori
J
J
Kasami
M
M
Utsumi
M
M
Kaneda
T
T
D000163:Acquired Immunodeficiency Syndrome / Q000175:diagnosis*; D000328:Adult; D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D002384:Catalysis; D002460:Cell Line; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D008297:Male; D008875:Middle Aged; D015341:Nucleic Acid Probes; D016612:Paraffin Embedding; D020135:Peptide Nucleic Acids
D015341:Nucleic Acid Probes; D020135:Peptide Nucleic Acids