Design of polymerase chain reaction primers for the selective amplification of HIV-1 RNA in the presence of HIV-1 DNA

Tobias R. Kollmann, Xiajun Zhuangt, Arye Rubinstein, Harris Goldstein

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Objective: To develop a sensitive method for the specific detection of HIV-1 RNA. Design: Following reverse transcription, the presence of HIV-1 RNA can be detected by polymerase chain reaction (PCR) amplification. Since specific detection of HIV-1 RNA may be complicated by contamination with minute quantities of HIV-1 DNA, samples are treated with deoxyribonuclease (DNase) prior to analysis. This additional step increases the possibility of RNA degradation and sample contamination. Methods: A primer, HG141, was designed to hybridize to the poly(A) tract present in HIV-1 genomic and all HIV-1 messenger (m) RNA with its 5′ end and to the region upstream of the poly(A) tract with its 3′ end. The increased stability of the HG141 primer/HIV-1 RNA or complementary (c) DNA complex, enabled PCR amplification to be performed with HG141 and the return primer HG62 at an annealing temperature above the melting temperature (Tm) of the primer-HIV-1 DNA complex. Results: After reverse transcription of samples obtained from HIV-1-infected H9 cells, HG62/141-primed PCR amplification specifically detected HIV-1 RNA sequences without the need for DNase pre-treatment. This technique was more sensitive for the detection of HIV-1 RNA than SK38/39-primed PCR amplification of DNase-treated samples. Conclusions: Since the presence of HIV-1 RNA is indicative of HIV-1 replication for the presence of HIV-1 virions, the RNA-specific primer described should facilitate the assessment of HIV-1 replication and the plasma HIV-1 viral load in HIV-1-infected individuals. This should prove useful in the evaluation of the effects of therapeutic interventions on HIV-1 infection.

Original languageEnglish (US)
Pages (from-to)547-552
Number of pages6
JournalAIDS
Volume6
Issue number6
StatePublished - Jun 1992

Fingerprint

HIV-1
RNA
Polymerase Chain Reaction
DNA
Deoxyribonucleases
Poly A
Reverse Transcription
Temperature
RNA Stability
Therapeutic Uses
Viral Load
Virion
Freezing
HIV Infections

Keywords

  • DNA replication
  • Gene amplification
  • Polymerase chain reaction
  • Viral DNA analysis
  • Viral RNA analysis

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

Design of polymerase chain reaction primers for the selective amplification of HIV-1 RNA in the presence of HIV-1 DNA. / Kollmann, Tobias R.; Zhuangt, Xiajun; Rubinstein, Arye; Goldstein, Harris.

In: AIDS, Vol. 6, No. 6, 06.1992, p. 547-552.

Research output: Contribution to journalArticle

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abstract = "Objective: To develop a sensitive method for the specific detection of HIV-1 RNA. Design: Following reverse transcription, the presence of HIV-1 RNA can be detected by polymerase chain reaction (PCR) amplification. Since specific detection of HIV-1 RNA may be complicated by contamination with minute quantities of HIV-1 DNA, samples are treated with deoxyribonuclease (DNase) prior to analysis. This additional step increases the possibility of RNA degradation and sample contamination. Methods: A primer, HG141, was designed to hybridize to the poly(A) tract present in HIV-1 genomic and all HIV-1 messenger (m) RNA with its 5′ end and to the region upstream of the poly(A) tract with its 3′ end. The increased stability of the HG141 primer/HIV-1 RNA or complementary (c) DNA complex, enabled PCR amplification to be performed with HG141 and the return primer HG62 at an annealing temperature above the melting temperature (Tm) of the primer-HIV-1 DNA complex. Results: After reverse transcription of samples obtained from HIV-1-infected H9 cells, HG62/141-primed PCR amplification specifically detected HIV-1 RNA sequences without the need for DNase pre-treatment. This technique was more sensitive for the detection of HIV-1 RNA than SK38/39-primed PCR amplification of DNase-treated samples. Conclusions: Since the presence of HIV-1 RNA is indicative of HIV-1 replication for the presence of HIV-1 virions, the RNA-specific primer described should facilitate the assessment of HIV-1 replication and the plasma HIV-1 viral load in HIV-1-infected individuals. This should prove useful in the evaluation of the effects of therapeutic interventions on HIV-1 infection.",
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