Two-step real-time PCR quantification of all subtypes of human immunodeficiency virus type 1 by an in-house method using locked nucleic acid-based probes.

Current HIV-1 viral-load assays are too expensive and time-consuming for small sample quantity or resource-limited setting. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes. We developed an internally controlled, two-step, reverse transcription-initiated real-time PCR protocol on the LightCycler instrument achieving a favourable detection limit with an extended quantification range, detecting all HIV-1 subtypes suitable for laboratories with low sample throughput. The detection limit was found to be 100 copies/ml, the dynamic range up to 500,000,000 copies/ml. Intra and inter assay imprecision were 1.6% and 2.0% (n=10), respectively. The assay was calibrated against WHO Standard 97/656. The HIV-1 RNA values obtained by real-time PCR assay were highly correlated with those obtained by the Cobas Amplicor HIV-1 Monitor (r = 0.954; p < 0.001). This rapid (3 h) and cost effective assay is suitable for both HIV-1 detection and disease monitoring with the ability to detect and quantify all HIV-1 subtypes including O1 and O2.