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Updated: Jun 18, 2026
Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells
Published on: January 30, 2019
Improvement of an ultrasensitive human immunodeficiency virus type 1 real-time reverse transcriptase-polymerase chain
Anke Edelmann1, Ulrich Kalus, Anke Oltmann
1Institute of Virology Helmut-Ruska-Haus, Charité University Medicine Berlin, Berlin, Germany. anke.edelmann@charite.de
Background:
Human immunodeficiency virus Type 1 (HIV-1) assays applying nucleic acid testing (NAT) rely on HIV-1 sequence-specific primers and probes. Their hybridization can be limited or abolished by genetic polymorphisms occurring in the target sequence.
Study Design And Methods:
Blood donations are routinely tested for HIV-1/2 antibodies and for HIV-1 RNA in our blood transfusion unit. Recently, HIV-1 RNA was undetectable with an established in-house real-time long terminal repeat (LTR) reverse transcriptase-polymerase chain reaction (RT-PCR) in two cases, whereas serologic assays were positive. The reason for this discrepancy was elucidated by sequencing of the NAT target region in the respective single donations. An improved primer was designed and tested on HIV-1 reference panels and blood donations to ensure reliable detection of HIV-1 RNA.
Results:
Direct sequencing of the target region, isolated from samples of two unrelated HIV-positive blood donors, revealed one and four mismatches in the hybridization domain of the forward primer, respectively. Both viruses belong to HIV-1 Subtype B. LTR RT-PCR with an additional forward primer was suitable for all strains of HIV-1 tested with high sensitivity.
Conclusions:
Surveillance of HIV-1 genetic diversity is essentially required to continually evaluate its impact on performance of diagnostic and patient monitoring assays.
