Engineering Insights for Multiplexed Real-Time Nucleic Acid Sequence-Based Amplification (NASBA): Implications for Design of Point-of-Care Diagnostics
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.
We offer a systematic analysis of NASBA design with a view toward expanding its applicability.
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.
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 (t1/2) 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 t1/2 by 10 min. Finally, we confirm the efficacy of two molecular beacon design schemes and discuss their multiplexing utility in two clinical scenarios.
This study provides a pathway for using NASBA in developing POC diagnostic assays.
- 5.Chantratita W, Pongtanapisit W, Piroj W, Srichunrasmi C, Seesuai S. Development and comparison of the real-time amplification based methods—NASBA-Beacon, RT-PCR taqman and RT-PCR hybridization probe assays–for the qualitative detection of sars coronavirus. Southeast Asian J Trop Med Public Health. 2004;35(3):623–9. (Epub 2005/02/04).PubMedGoogle Scholar
- 9.Vandamme AM, Schmit JC, Van Dooren S, Van Laethem K, Gobbers E, Kok W, et al. Quantification of HIV-1 RNA in plasma: comparable results with the NASBA HIV-1 RNA QT and the AMPLICOR HIV monitor test. J Acquir Immune Defic Syndr Hum Retrovirol. 1996;13(2):127–39. (Epub 1996/10/01).PubMedCrossRefGoogle Scholar
- 12.Dyer JR, Gilliam BL, Eron JJ, Grosso L, Cohen MS, Fiscus SA. Quantitation of human immunodeficiency virus type 1 RNA in cell free seminal plasma: comparison of NASBA™ with Amplicor™ reverse transcription-PCR amplification and correlation with quantitative culture. J Virol Methods. 1996;60(2):161–70.PubMedCrossRefGoogle Scholar
- 18.Markham NR. DI-Nucleic Acid hybridization and melting prediction. The UNAFold Web Server. University at Albany: The RNA Institute College of Arts and Sciences. 2011. http://mfold.rna.albany.edu/?q=dinamelt. Accessed June 2011.
- 19.Technologies L. The Basics: in vitro transcription (cited 2011). http://www.invitrogen.com/site/us/en/home/References/Ambion-Tech-Support/probe-labeling-systems/general-articles/the-basics-in-vitro-transcription.html.