Engineering insights for multiplexed real-time nucleic acid sequence-based amplification (NASBA): implications for design of point-of-care diagnostics.

backgroundNucleic acid sequence-based amplification (NASBA) offers huge potential for low-cost, point-of-care (POC) diagnostic devices, but has been limited by high false-positive rates and the challenges of primer design.

objectiveWe offer a systematic analysis of NASBA design with a view toward expanding its applicability.

methodsWe examine the parameters that effect dimer formations, and we provide a framework for designing NASBA primers that will reduce false-positive results and make NASBA suitable for more POC diagnostic applications. Then we compare three different oligonucleotide sets to examine (1) the inhibitory effect of dimer formations, (2) false positives with poorly designed primers, and (3) the effect of beacon target location during real-time NASBA. The required T7 promoter sequence adversely affects the reaction kinetics, although the common abridged sequence can improve kinetics without sacrificing accuracy.

resultsWe demonstrate that poorly designed primers undergo real-time exponential amplification in the absence of target RNA, resulting in false positives with a time to half of the peak value (t(1/2)) of 50 min compared to 45 min for true positives. Redesigning the oligonucleotides to avoid inhibitory dimers eliminated false positives and reduced the true positive t(1/2) by 10 min. Finally, we confirm the efficacy of two molecular beacon design schemes and discuss their multiplexing utility in two clinical scenarios.

conclusionThis study provides a pathway for using NASBA in developing POC diagnostic assays.