Engineering Insights for Multiplexed Real-Time Nucleic Acid Sequence-Based Amplification (NASBA): Implications for Design of Point-of-Care Diagnostics
Rent the article at a discountRent now
* Final gross prices may vary according to local VAT.Get Access
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 (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.
This study provides a pathway for using NASBA in developing POC diagnostic assays.
- Compton J. Nucleic-acid sequence-based amplification. Nature. 1991;350(6313):91–2. CrossRef
- Collins RA, Ko LS, So KL, Ellis T, Lau LT, Yu AC. Detection of highly pathogenic and low pathogenic avian influenza subtype H5 (Eurasian lineage) using NASBA. J Virol Methods. 2002;103(2):213–25. (Epub 2002/05/15). CrossRef
- Collins RA, Ko LS, Fung KY, Lau LT, Xing J, Yu AC. A method to detect major serotypes of foot-and-mouth disease virus. Biochem Biophys Res Commun. 2002;297(2):267–74. (Epub 2002/09/19). CrossRef
- Lau LT, Reid SM, King DP, Lau AM, Shaw AE, Ferris NP, et al. Detection of foot-and-mouth disease virus by nucleic acid sequence-based amplification (NASBA). Vet Microbiol. 2008;126(1–3):101–10. (Epub 2007/08/31). CrossRef
- 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).
- Keightley MC, Sillekens P, Schippers W, Rinaldo C, George KS. Real-time NASBA detection of SARS-associated coronavirus and comparison with real-time reverse transcription-PCR. J Med Virol. 2005;77(4):602–8. (Epub 2005/10/29). CrossRef
- Lanciotti RS, Kerst AJ. Nucleic acid sequence-based amplification assays for rapid detection of West Nile and St. Louis encephalitis viruses. J Clin Microbiol. 2001;39(12):4506–13. CrossRef
- Bohmer A, Schildgen V, Lusebrink J, Ziegler S, Tillmann RL, Kleines M, et al. Novel application for isothermal nucleic acid sequence-based amplification (NASBA). J Virol Methods. 2009;158(1–2):199–201. (Epub 2009/05/12). CrossRef
- 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). CrossRef
- Vandamme AM, Van Dooren S, Kok W, Goubau P, Fransen K, Kievits T, et al. Detection of HIV-1 RNA in plasma and serum samples using the NASBA amplification system compared to RNA-PCR. J Virol Methods. 1995;52(1–2):121–32. CrossRef
- Schneider P, Wolters L, Schoone G, Schallig H, Sillekens P, Hermsen R, et al. Real-time nucleic acid sequence-based amplification is more convenient than real-time PCR for quantification of Plasmodium falciparum. J Clin Microbiol. 2005;43(1):402–5. (Epub 2005/01/07). CrossRef
- 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. CrossRef
- Luebke KJ, Balog RP, Garner HR. Prioritized selection of oligodeoxyribonucleotide probes for efficient hybridization to RNA transcripts. Nucl Acids Res. 2003;31(2):750–8. CrossRef
- Guatelli JC, Whitfield KM, Kwoh DY, Barringer KJ, Richman DD, Gingeras TR. Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc Natl Acad Sci. 1990;87(5):1874–8. CrossRef
- Deiman B, van Aarle P, Sillekens P. Characteristics and applications of nucleic acid sequence-based amplification (NASBA). Mol Biotechnol. 2002;20(2):163–79. CrossRef
- McCalla SE, Ong C, Sarma A, Opal SM, Artenstein AW, Tripathi A. A simple method for amplifying RNA targets (SMART). J Mol Diagn. 2012;14(4):328–35. CrossRef
- Ong C, Tai W, Sarma A, Opal SM, Artenstein AW, Tripathi A. Ligation with Nucleic Acid Sequence-Based Amplification. J Mol Diagn. 2012;14(3):206–13. CrossRef
- 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.
- 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.
- Owczarzy R, Moreira BG, You Y, Behlke MA, Walder JA. Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations. Biochemistry-Us. 2008;47(19):5336–53. CrossRef
- Engineering Insights for Multiplexed Real-Time Nucleic Acid Sequence-Based Amplification (NASBA): Implications for Design of Point-of-Care Diagnostics
Molecular Diagnosis & Therapy
Volume 17, Issue 3 , pp 185-192
- Cover Date
- Print ISSN
- Online ISSN
- Springer International Publishing AG
- Additional Links
- Industry Sectors
- Author Affiliations
- 1. Center for Biomedical Engineering, Brown University, Providence, RI, USA
- 2. School of Engineering and Division of Biology and Medicine, and the Warren Alpert School of Medicine, Brown University, Providence, RI, USA