PNA-in situ hybridization method for detection of HIV-1 DNA in virus-infected cells and subsequent detection of cellular and viral proteins.
We describe in situ hybridization protocols using peptide nucleic acid (PNA) as a probe for detecting HIV-1 DNA in virus-infected cells and the subsequent detection of cellular and/or viral proteins. Because a PNA probe of approx 20 bases was sufficiently long to detect a specific target sequence, a conserved sequence of such a short length was easily identified. Therefore, this probe is valuable even to identify quasi-species of HIV-1. In addition, we adopted a catalyzed signal amplification method to amplify weak viral DNA signals; thus, stringent washing was crucial for eliminating false-positive signals. Our double-staining method using PNA-in situ hybridization and subsequent immunostaining enabled the active and inactive proviruses to be distinguished.
Hagiwara
Tomoko
T
Clinical Research Center, National Hospital Organization Nagoya Medical Center, Aichi, Japan.
Hattori
Junko
J
Kaneda
Tsuguhiro
T
D015496:CD4-Positive T-Lymphocytes / Q000821:virology; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D016655:HIV Core Protein p24 / Q000032:analysis* / Q000235:genetics; D015658:HIV Infections / Q000175:diagnosis / Q000821:virology; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006684:HLA-DR Antigens / Q000032:analysis / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D020135:Peptide Nucleic Acids; D011533:Proviruses / Q000235:genetics; D014764:Viral Proteins / Q000032:analysis* / Q000235:genetics
D004279:DNA, Viral; D016655:HIV Core Protein p24; D006684:HLA-DR Antigens; D020135:Peptide Nucleic Acids; D014764:Viral Proteins