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

Abstract

Background: Nucleic 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.

Objective: We offer a systematic analysis of NASBA design with a view toward expanding its applicability.

Methods: We 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.

Results: We 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.

Conclusion: This study provides a pathway for using NASBA in developing POC diagnostic assays.

Fig. 1

Traditional NASBA begins with a non-cyclic phase, which creates the RNA product (RNA(−)). T7 polymerase uses the dsDNA from the non-cyclic phase as a template to transcribe RNA(−), which initiates the cyclic phase. RNA(−) is the product and template of the cyclic amplification phase and can be detected by end-point gel electrophoresis or in real-time NASBA using molecular beacons. The T7 promoter sequence must be incorporated at the 5′ end of primer 1 (as shown in the cyclic phase). The underlined sequence represents the abridged sequence, and the +1 base is the first base incorporated into the RNA during transcription

Fig. 2

Molecular beacon designs. a Beacon design (i) is designed for multiplexing. A variety of probes can be used to target a group of individual sequences, all of which are indicated with one molecular beacon. b Beacon design (ii) is designed for targeting a specific sequence rather than targeting a group of sequences

Fig. 3

Primer dimer theoretical hybridization energies simulated with DINAMelt. Energy rules: DNA @ 41 °C, C _T = 10 μM, [Na⁺] = 73.5 mM, [Mg⁺⁺] = 12.5 mM. a Primer alone b primer with abridged T7 promoter c primer with full T7 promoter

Fig. 4

Real-time NASBA of poorly designed HIV-1 K103N oligonucleotide set (Old) vs. redesigned HIV-1 K103N set (New) vs. H3 influenza oligonucleotide set. 1nM probes used and no probe controls included. Standard deviation was within 5 % of results (error bars not shown)

Fig. 5

a T7 polymerization of unexpected P1-P1 RNA. There are two locations for the T7 polymerase to polymerize RNA since the T7 promoter exists on both strands 5′ → 3′. b T7 polymerization of expected P1-Probe RNA