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G. Shaw, B. Hahn, S. Arya, J. Groopman, R. Gallo, F. Wong-Staal (1984)
Molecular characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deficiency syndrome.Science, 226 4679
Elisabeth Stoeckl, N. Barrett, F. Heinz, M. Banekovich, G. Stingl, Klaus Guggenberger, F. Dorner, C. Kunz (1989)
Efficiency of the polymerase chain reaction for the detection of human immunodeficiency virus type (HIV‐1) DNA in the lymphocytes of infected persons: Comparison to antigen‐enzyme‐linked immunosorbent assay and virus isolationJournal of Medical Virology, 29
M. Harper, L. Marselle, R. Gallo, F. Wong-Staal (1986)
Detection of lymphocytes expressing human T-lymphotropic virus type III in lymph nodes and peripheral blood from infected individuals by in situ hybridization.Proceedings of the National Academy of Sciences of the United States of America, 83 3
G Yang, R Pu, A Comeau, GN Vyas (1994)
Flow cytometric immunodetection of PCR‐amplified HIV‐1 proviral DNA in infants born to seropositive mothers, 102
D. Kemp, D. Smith, S. Foote, N. Samaras, M. Peterson (1989)
Colorimetric detection of specific DNA segments amplified by polymerase chain reactions.Proceedings of the National Academy of Sciences of the United States of America, 86 7
G. Yang, S. Garhwal, J. Olson, G. Vyas (1994)
Flow cytometric immunodetection of human immunodeficiency virus type 1 proviral DNA by heminested PCR and digoxigenin-labeled probesClinical Diagnostic Laboratory Immunology, 1
M. Rogers, C. Ou, M. Rayfield, P. Thomas, E. Schoenbaum, E. Abrams, K. Krasinski, P. Selwyn, Jennifer Moore, A. Kaul, K. Grimm, M. Bamji, G. Schochetman (1989)
Use of the polymerase chain reaction for early detection of the proviral sequences of human immunodeficiency virus in infants born to seropositive mothersInternational Journal of Gynecology & Obstetrics, 31
MP Busch, BA Eble, H Khayam‐Bashi, E Murphy, D Heilbron, S Kwok, J Sninsky, HA Perkins, GN Vyas (1991)
Evaluation of contemporary screened blood donors for occult HIV‐1 infection using pooled cell culture and DNA amplification techniques, 325
JH Kenten, J Casadel, J Link, S Lupold, J Willey, M Powell, A Rees, R Massey (1989)
Rapid electrochemiluminescence assay of polymerase chain reaction products, 86
G Yang, PP Ulrich, RA Aiyer, BD Rawal, GN Vyas (1993)
Detection of hepatitis B virus in plasma using flow cytometric analyses of PCR‐amplified DNA incorporating digoxigenin‐11‐dUTP, 81
B. Schmidt (1991)
A rapid chemiluminescence detection method for PCR-amplified HIV-1 DNA.Journal of virological methods, 32 2-3
KA Clouse (1989)
Monokine regulation of human immunodeficiency virus ‐1 expression in a chronically infected, 142
ME Harper, LM Marselle, RC Gallo, F Wong‐Staal (1986)
Detection of lymphocytes expressing HTLV‐III in lymph nodes and peripheral blood from infected individuals by in situ hybridization, 83
P. Dahlen, A. Iitiä, G. Skagius, Å. Frostell, M. Nunn, M. Kwiatkowski (1991)
Detection of human immunodeficiency virus type 1 by using the polymerase chain reaction and a time-resolved fluorescence-based hybridization assayJournal of Clinical Microbiology, 29
J. Edwards, P. Ulrich, P. Weintrub, M. Cowan, J. Levy, D. Wara, G. Vyas (1989)
Polymerase chain reaction compared with concurrent viral cultures for rapid identification of human immunodeficiency virus infection among high-risk infants and children.The Journal of pediatrics, 115 2
Kazuo Suzuki, Naoaki Okamoto, S. Watanabe, Tokio Kano (1992)
Chemiluminescent microtiter method for detecting PCR amplified HIV-1 DNA.Journal of virological methods, 38 1
Michael Busch, Bernard Eble, Hassan Khayam-Bashi, David Heilbron, Edward Murphy, Shirley Kwok, J. Sninsky, Herbert Perkins, Girish Vyas (1991)
Evaluation of screened blood donations for human immunodeficiency virus type 1 infection by culture and DNA amplification of pooled cells.The New England journal of medicine, 325 1
K. Clouse, D. Powell, I. Washington, Guido Poli, K. Strebel, W. Farrar, P. Barstad, J. Kovacs, A. Fauci, T. Folks (1989)
Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone.Journal of immunology, 142 2
C. Ou, S. Kwok, S. Mitchell, D. Mack, J. Sninsky, J. Krebs, P. Feorino, D. Warfield, G. Schochetman (1988)
DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells.Science, 239 4837
J. Scillian, T. McHugh, M. Busch, M. Tam, M. Fulwyler, D. Chien, G. Vyas (1989)
Early detection of antibodies against rDNA-produced HIV proteins with a flow cytometric assay.Blood, 73 7
GN Vyas, G Yang, E Murphy (1994)
Transfusion‐related transmissible diseases: Detection by PCR‐amplified genes of the microbial agents, 8
Girish Vyas, Gang Yang, E. Murphy (1994)
Transfusion-related transmissible diseases: detection by polymerase chain reaction-amplified genes of the microbial agents.Transfusion medicine reviews, 8 4
G. Yang, P. Ulrich, RA Aiyer, BD Rawal, G. Vyas (1993)
Detection of hepatitis B virus in plasma using flow cytometric analyses of polymerase chain reaction-amplified DNA incorporating digoxigenin-11-dUTP.Blood, 81 4
J. Guatelli, T. Gingeras, Douglas Richman (1989)
Nucleic acid amplification in vitro: detection of sequences with low copy numbers and application to diagnosis of human immunodeficiency virus type 1 infectionClinical Microbiology Reviews, 2
B. Patterson, M. Till, P. Otto, C. Goolsby, M. Furtado, L. Mcbride, Steven Wolinsky (1993)
Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry.Science, 260 5110
C. Hart, T. Spira, Jennifer Moore, J. Sninsky, G. Schochetman, A. Lifson, J. Galphin, C. Ou (1988)
DIRECT DETECTION OF HIV RNA EXPRESSION IN SEROPOSITIVE SUBJECTSThe Lancet, 332
M. Loche, B. Mach (1988)
IDENTIFICATION OF HIV-INFECTED SERONEGATIVE INDIVIDUALS BY A DIRECT DIAGNOSTIC TEST BASED ON HYBRIDISATION TO AMPLIFIED VIRAL DNAThe Lancet, 332
Serological assays are routinely used in the laboratory diagnosis of human immunodeficiency virus type‐1 (HIV‐1) infection, but the polymerase chain reaction (PCR) is ultimately the most sensitive and direct method for establishing definitive diagnosis. As an alternative to the conventional radioactive PCR procedure we have developed and evaluated a pair of rapid nonradioisotopic flow cytometric detection methods. Using heminested PCR we directly incorporated fluorescein‐12‐dUTP (fluo‐dUTP) or digoxigenin‐11‐dUTP (dig‐dUTP) into the PCR‐amplicons. The labeled amplicons were hybridized with biotinylated antisense and sense probes, followed by capture of the hybrid DNA using streptavidin‐coated beads which were finally analyzed in a flow cytometer by (1) direct detection of the fluorescence intensity of the amplicons incorporating fluo‐dUTP and (2) immunodetection of the amplicons incorporating dig‐dUTP by anti‐digoxigenin IgG labeled with fluorescein isothiocyanate (FITC). Although both assays were functionally comparable with radiolabeled probe in reliably detecting as low as five copies of HIV‐1 proviral DNA sequences, the immunodetection of dig‐dUTP consistently yielded higher mean channel fluorescence and gave a stable signal over an extended period of 12–14 weeks. In testing a panel of 20 pedigreed PBMC specimens from blood donors with or without HIV‐1 infection, the results of both flow cytometric assays were identical with those of the conventional radioactive procedure. Therefore, we conclude that the dig‐dUTP incorporation in amplicons, hybridization with a pair of sense‐antisense biotinylated probes and immunodetection of hybrids by flow cytometric analyses is the nonisotopic method of choice for PCR‐diagnosis of HIV‐1 infection. © 1995 Wiley‐Liss, Inc.
Cytometry Part A – Wiley
Published: Oct 1, 1995
Keywords: ; ; ; ; ; ;
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