title: 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.
  
  
  CONCLUSIONS
  This study provides a pathway for using NASBA in developing POC diagnostic assays.
authors.affiliation: Center for Biomedical Engineering, Brown University, Providence, RI, USA.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Morabito
authors.forename: Clay
authors.initials: C
authors.lastname: Wiske
authors.forename: Anubhav
authors.initials: A
authors.lastname: Tripathi
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000096:biosynthesis* / Q000235:genetics; D005189:False Positive Reactions; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D019095:Point-of-Care Systems; D021161:Self-Sustained Sequence Replication / Q000379:methods*
chemical_list: D017931:DNA Primers