Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR

Abstract

Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually depend on exonuclease activity of DNA polymerase (e.g. TaqMan probe) or oligonucleotide hybridization (e.g. molecular beacon). Probe design often needs to be specifically tailored either to tolerate or to differentiate between sequence variations. The conventional probe technologies offer limited flexibility to meet these diverse requirements. Here, we introduce a novel partially double-stranded linear DNA probe design. It consists of a hybridization probe 5′-labeled with a fluorophore and a shorter quencher oligo of complementary sequence 3′-labeled with a quencher. Fluorescent signal is generated when the hybridization probe preferentially binds to amplified targets during PCR. This novel class of probe can be thermodynamically modulated by adjusting (i) the length of hybridization probe, (ii) the length of quencher oligo, (iii) the molar ratio between the two strands and (iv) signal detection temperature. As a result, pre-amplification signal, signal gain and the extent of mismatch discrimination can be reliably controlled and optimized. The applicability of this design strategy was demonstrated in the Abbott RealTime HIV-1 assay.

Immiscible Phase Nucleic Acid Purification Eliminates PCR Inhibitors with a Single Pass of Paramagnetic Particles through a Hydrophobic Liquid

“…The eluted RNA was quantified by RT-qPCR using the Abbott RealTime HIV-1 Amplification Reagent Kit (Abbott Molecular, Des Plaines, IL). 19 No statistically significant differences were observed between the C q values for the samples purified using the liquid wax and the manual protocol (two-sided P values of 0.76, 0.99, and 0.40, respectively), demonstrating that the movement of PMPs through the wax does not interfere with the binding of the NA to the PMPs or inhibit the subsequent amplification by RT-qPCR (Figure 2A). Of particular interest was that the alcohol evaporation step required by the Ambion protocol could be eliminated.…”

“…The PMPs were aggregated and removed from the elution buffer. HIV-1 viral load quantification was performed for each sample using the Abbott RealTime HIV-1 Amplification Reagent Kit 19 …”

“…Lysis buffer (15 l) and 200 l of alcohol wash buffer were added to the solution and the IPF purification was performed as before. The purified DNA from each sample was amplified using the Abbott RealTime HIV-1 Amplification Reagent Kit (Abbott Molecular, Des Plaines, IL) 19 in 25-l reaction volume. Amplification reactions were performed in Cepheid SmartCycler II (Sunnyvale, CA).…”

Evaluation of the Abbott Investigational Use Only RealTime HIV-1 Assay and Comparison to the Roche Amplicor HIV-1 Monitor Test, Version 1.5

“…The frequency of observed polymorphisms was 0.2%, 0.6%, and 1.2% respectively, for the forward primer, probe, and reverse primer, respectively. 11 One of the polymorphic positions in the forward primer (Q148) is associated with integrase inhibitor resistance. A systematic analysis using engineered transcripts containing all possible resistance-associated substitutions at codon 148 revealed no discernible impact on assay performance.…”

“…6 -8,10 The primers and probe system targeting the integrase region of the HIV-1 genome were recently described. 11 Versant HIV-1 RNA 3.0 Assay (bDNA)…”