Detection of HIV DNA and RNA Using PCR

Susan Moir1, Tae‐Wook Chun1

1 National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 12.6
DOI:  10.1002/0471142735.im1206s42
Online Posting Date:  May, 2001
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Abstract

This extensively updated unit describes the detection of HIV DNA and RNA using PCR using two basic techniques for quantifying the levels of viral DNA and RNA in infected cells. The schemes for both techniques include isolation of nucleic acids, PCR reactions and detection of amplified products using Southern blotting.

     
 
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Table of Contents

  • Basic Protocol 1: Amplification and Detection of HIV DNA Using PCR (DNA‐PCR)
  • Basic Protocol 2: Detection of HIV RNA by Reverse Transcriptase‐PCR (RT‐PCR)
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Amplification and Detection of HIV DNA Using PCR (DNA‐PCR)

  Materials
  • HIV‐infected cells to be tested for the presence of HIV DNA
  • HIV‐negative cells
  • ACH‐2 T cell line (AIDS Research and Reference Reagent Program, # 349)
  • DNA isolation kit (Puregene, Gentra Systems)
  • Isopropanol
  • 70% ethanol
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 20 mg/ml proteinase K
  • recipeCell lysis buffer (used only for step ; see recipe)
  • HIV‐specific PCR primers (Table 12.6.1)
  • 10 mM dNTP mix (Perkin‐Elmer; unit 10.20)
  • recipe10× PCR buffer (see recipe)
  • 50 mM MgCl 2
  • 2.5 U Platinum Taq DNA polymerase (Life Technologies)
  • 40 pmol oligonucleotide probe (Table 12.6.1)
  • 5× T4 polynucleotide kinase forward reaction buffer (provided with T4 polynucleotide kinase)
  • 10 U/µl T4 polynucleotide kinase (Life Technologies)
  • 10 µCi/µl [γ‐32P]ATP
  • 1.8% (w/v) agarose gel containing 0.5 µg/ml ethidium bromide
  • 0.5×, 1×, and 2× SSC containing 0.1% (w/v) SDS
  • recipeHybridization solution (see recipe)
  • 10 mg/ml salmon sperm DNA (Life Technologies)
  • 56° and 94°C water baths
  • 8‐tube strip MicroAmp optical tubes (PE Biosystems)
  • 8‐cap strip MicroAmp optical caps (PE Biosystems)
  • Thermal cycler (e.g., GeneAmp PCR System 9700, Perkin‐Elmer)
  • 0.5‐ and 1.5‐ml microcentrifuge tubes
  • Elutip‐D columns (Schleicher & Schuell)
  • Nylon membrane (Zeta Probe GT, Bio‐Rad)
  • Chromatography paper (Whatman)
  • UV cross‐linker (Stratagene)
  • Glass hybridization tube (Stovall)
  • Hybridization oven (Stovall)
  • Plastic dish with tight cover
  • Phosphor imager system, including screen, image eraser and Storm 860 (Molecular Dynamics)
  • Image Quant software (Molecular Dynamics)
  • Additional reagents and equipment for culture of T cells (unit 12.1), counting cells ( appendix 3A), isolation of mononuclear cells (unit 12.2), concentration and purification of DNA with Elutip‐D columns (unit 10.5), Southern blot hybridization (unit 10.7), autoradiography ( appendix 3J), and agarose gel electrophoresis (unit 10.4)

Basic Protocol 2: Detection of HIV RNA by Reverse Transcriptase‐PCR (RT‐PCR)

  Materials
  • SP6 in vitro transcription kit (e.g., MEGAscript, Ambion)
  • Control plasmid (SP64AH2) to transcribe unspliced form of HIV RNA
  • DEPC‐treated H 2O (unit 12.8)
  • recipeLithium chloride precipitation solution (see recipe)
  • 70% and 75% ethanol
  • HIV‐infected cells
  • TRIzol reagent (Life Technologies)
  • Chloroform
  • Isopropanol
  • 500 mM EDTA, pH 8.0 ( appendix 2A)
  • 10 mM 4dNTP mix (Life Technologies)
  • 50 ng/µl random hexamers (Life Technologies)
  • recipe10× RT‐PCR buffer (see recipe)
  • 25 mM MgCl 2
  • 1.5 M DTT (unit 12.8)
  • 40 U/µl recombinant ribonuclease inhibitor (e.g., RNaseOUT, Life Technologies)
  • 50 U/µl Superscript II reverse transcriptase (Life Technologies)
  • 2 U/µl RNase H (Life Technologies)
  • 5 µM 5′ primer
  • 5 µM 3′ primer
  • 2.5 U Platinum Taq DNA polymerase
  • 0.5‐ and 1.5 ml microcentrifuge tubes
  • 25°, 37°, 65°, and 95°C water baths
  • 8‐tube strip MicroAmp optical tubes (PE Biosystems)
  • 8‐cap strip MicroAmp optical caps (PE Biosystems)
  • Thermal cycler (e.g., GeneAmp PCR System 9700, Perkin‐Elmer)
NOTE: Precautions must be taken to avoid the risk of RNA degradation by ribonuclease contamination. These include the use of RNase‐free sterile plasticware and solutions, the addition of an RNase inhibitor during the RT reaction, and the use of DEPC‐treated water in the preparation of RNA transcripts and cDNA templates. To avoid DNA contamination during the PCR, the RNA transcripts are pre‐treated with DNase.
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Figures

Videos

Literature Cited

Literature Cited
   Chun, T.W., Carruth, L., Finzi, D., Shen, X., DiGiuseppe, J.A., Taylor, H., Hermankova, M., Chadwick, K., Margolick, J., Quinn, T.C., Kuo, Y.H., Brookmeyer, R., Zeiger, M.A., Barditch‐Crovo, P., and Siliciano, R.F. 1997. Quantification of latent tissue reservoirs and total body viral load in HIV‐1 infection. Nature 387:183‐188.
   Delwart, E.L., Shpaer, E.G., Louwagie, J., McCutchan, F.E., Grez, M., Rubsamen‐Waigmann, H., and Mullins, J.I. 1993. Genetic relationships determined by a DNA heteroduplex mobility assay: Analysis of HIV‐1 env genes. Science 262:1257‐1261.
   Ho, D.D., Neumann, A.U., Perelson, A.S., Chen, W., Leonard, J.M., and Markowitz, M. 1995. Rapid turnover of plasma virions and CD4 lymphocytes in HIV‐1 infection. Nature 373:123‐126.
   Lewin, S.R., Vesanen, M., Kostrikis, L., Hurley, A., Duran, M., Zhang, L., Ho, D.D., and Markowitz, M. 1999. Use of real‐time PCR and molecular beacons to detect virus replication in human immunodeficiency virus type 1‐infected individuals on prolonged effective antiretroviral therapy. J. Virol. 73:6099‐6103.
   Ou, C.Y., Kwok, S., Mitchell, S.W., Mack, D.H., Sninsky, J.J., Krebs, J.W., Feorino, P., Warfield, D., and Schochetman, G. 1988. DNA amplification for direct detection of HIV‐1 in DNA of peripheral blood mononuclear cells. Science 239:295‐297.
   Saksela, K., Muchmore, E., Girard, M., Fultz, P., and Baltimore, D. 1993. High viral load in lymph nodes and latent human immunodeficiency virus (HIV) in peripheral blood cells of HIV‐1‐infected chimpanzees. J. Virol. 67:7423‐7427.
   Vesanen, M., Markowitz, M., Cao, Y., Ho, D.D., and Saksela, K. 1997. Human immunodeficiency virus type‐1 mRNA splicing pattern in infected persons is determined by the proportion of newly infected cells. Virology 236:104‐109.
   Wei, X., Ghosh, S.K., Taylor, M.E., Johnson, V.A., Emini, E.A., Deutsch, P., Lifson, J.D., Bonhoeffer, S., Nowak, M.A., Hahn, B.H., and et al. 1995. Viral dynamics in human immunodeficiency virus type 1 infection. Nature 373:117‐122.
   Zack, J.A., Arrigo, S.J., Weitsman, S.R., Go, A.S., Haislip, A., and Chen, I.S. 1990. HIV‐1 entry into quiescent primary lymphocytes: molecular analysis reveals a labile, latent viral structure. Cell 61:213‐222.
Internet Resources
  http://www.niaid.nih.gov/daids/vir_manual
  Provides many detailed techniques for those who wish to use commercial kits to determine HIV RNA levels in plasma samples and HIV proviral DNA levels in cells.
  http://www.aidsreagent.org
  Offers a wealth of HIV‐related reagents to the scientific community.
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