Real-time quantitative PCR and fast QPCR have similar sensitivity and accuracy with HIV cDNA late reverse transcripts and 2-LTR circles

Abstract

Real-time fluorescent quantitative PCR (universal QPCR) methods are used routinely in both academic and clinical research to measure HIV cDNA. Fast QPCR allows for faster ramping times between cycles and smaller reaction volumes, but may lose sensitivity and accuracy. We demonstrate that primer sets for HIV late reverse transcripts and 2-LTR circles have similar sensitivity and accuracy with either universal or fast QPCR methods. However, both cost and time are reduced with fast QPCR.

Fig. 1

QPCR primer sets and linear or circular HIV cDNA. HIV late reverse transcripts are measured by the primers MH531 and MH532. This amplicon spans the HIV primer binding site and is present in all forms of HIV cDNA, including linear cDNA, integrated provirus, 2-LTR circles, and 1-LTR circles. All complete reverse transcripts are measured by the primer set MH531 and MH532. 2-LTR circles are specifically amplified by the primers MH535 and KY268. The junction of U5 and U3 is unique to 2-LTR circles. The 2-LTR circle primer pair spans this unique junction in 2-LTR circles and will not amplify alternative HIV cDNA forms.

Fig. 2

Universal QPCR and fast QPCR amplification plots. (A) Universal QPCR amplification plots of HIV late reverse transcripts (left panel) and HIV 2-LTR circles (right panel). (B) Fast QPCR amplification plot of HIV late reverse transcripts (left panel) and HIV 2-LTR circles (right panel). Triplicate samples of 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, and 10⁸ target molecules were amplified for 40 cycles. The horizontal red line indicates the threshold determined by the SDS software.

Fig. 3

Standard curves from universal QPCR and fast QPCR. (A) Universal QPCR derived standard curves for HIV late reverse transcripts (left panel) and HIV 2-LTR circles (right panel). (B) Fast QPCR derived standard curves for HIV late reverse transcripts (left panel) and HIV 2-LTR circles (right panel). Triplicate samples of 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, and 10⁸ target molecules were amplified for 40 cycles. Best fit lines were generated by the SDS software. For all standard curves R² > 0.995.