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

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 (T_m) 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.