Background: Conventional real time PCR assays utilize Taqman technology where fluorescent signal is generated by cleavage of the Taqman probe by the exonuclease activity of the polymerase during primer extension. Mismatches between target and a Taqman probe may result in inefficient binding and cleavage during the relatively high temperature of primer extension resulting in under-estimation of viral load or false negative results. Considering the genetic diversity of HIV, a novel probe was designed for the RealTime  HIV-1^TM* assay to ensure tolerance to mismatches. The probe is partially double-stranded, and labeled with a fluorophore at the 5’end. The opposite shorter strand, complementary to the 5’ end of the probe, is labeled with a quencher at its 3’end. In the absence of HIV target, the fluorescence is quenched.  In the presence of HIV target, the probe preferentially hybridizes to the HIV target, and signal is generated. Since signal generation is not dependent on the exonuclease activity of the polymerase, primer extension step can be uncoupled from the signal generation/read step in PCR, thus allowing lowering the read temperature where mismatches between probe and HIV target are well tolerated and the unbound probe is quenched.

Methods: Tolerance to mismatches was evaluated by melt curve analysis between the HIV probe and targets with multiple mismatches, and by dilution linearity using 11 unique transcripts from Group M, O, and N isolates. Additionally the detection of diverse samples was evaluated with the WHO subtype panel and a subtype panel of 88 samples from Group M Subtypes A-H and Group O samples. The assay was also evaluated for sensitivity, specificity, precision and linearity.

Results: Multiple mismatches are well tolerated by the partially double stranded probe design. Dilution linearity for each transcript was demonstrated from 1 million copies/ml to 50 copies/ml. All samples in the subtype panels were detected.  25 copies/mL were detected with 95% probability, and assay specificity was 100 %. The linear range of the assay extends from 40 copies/mL to 10 million copies/mL.

Conclusion:  A novel partially double-stranded fluorescent probe design, and selection of  primers and probe from highly conserved regions of the pol gene allow the RealTime HIV-1 assay to quantitate diverse Group M subtypes A-H, Group O, and Group N samples. The assay is highly sensitive, specific, precise, and provides a wide dynamic range.

*Not available in the US