A novel real-time multiplex reverse transcriptase-polymerase chain reaction for the detection of HIV-1 RNA by using dual-specific armored RNA as internal control

Objective: Transmission of HIV-1 and diagnosis of infection in hospitals and public health settings remains a worldwide concern. HIV-1 detection is sometimes not possible using current commercial assays, probably due to mismatches between the selected primers and probes.

Methods: By screening primers and probes, we developed a dual-specificity probe real-time reverse transcriptase-polymerase chain reaction (DSPrtRT-PCR) assay using dual-specific armored RNA as the internal control. The specificity and sensitivity were compared between the monospecificity probe real-time and DSPrtRT-PCR techniques.

Results: The sensitivity of DSPrtRT-PCR improved significantly, with no effect on its specificity. The detection limit was 173 IU/ml. All the HIV-1 group M and group O could be detected. In clinical assays, 1,000 copies/ml of armored RNA was required as internal control. When applied to negative samples, 100% specificity was achieved. Among 60 samples from the tested patients, DSPrtRT-PCR demonstrated high sensitivity, accurately detecting 50 positives and 10 negatives that were confirmed by the COBAS AmpliScreen assay.

Conclusion: DSPrtRT-PCR is a more efficient and effective viral assay with high sensitivity and specificity as compared to monospecificity probe PCR. It can be widely applied in blood donor screening and qualitative individual detection of HIV-1 RNA.