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Title: Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA

Abstract

Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer-dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40-45 bases). Although primer-dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMP or RT-LAMP assays. In this study, we examine the impact of primer-dimers and hairpins on previously-published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter to predict the probability of non-specific amplification associated with LAMP primers.

Authors:
 [1];  [1];  [1];  [2];  [2]
  1. Sandia National Lab. (SNL-CA), Livermore, CA (United States)
  2. Univ. of Texas Medical Branch, Galveston, TX (United States)
Publication Date:
Research Org.:
Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)
Sponsoring Org.:
National Institutes of Health (NIH); USDOE
OSTI Identifier:
1429722
Report Number(s):
SAND-2017-13686J
Journal ID: ISSN 0003-2654; ANALAO; 659610
Grant/Contract Number:  
AC04-94AL85000
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Analyst
Additional Journal Information:
Journal Volume: 143; Journal Issue: 8; Journal ID: ISSN 0003-2654
Publisher:
Royal Society of Chemistry
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Meagher, Robert J., Priye, Aashish, Light, Yooli K., Huang, Cheng, and Wang, Eryu. Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA. United States: N. p., 2018. Web. doi:10.1039/c7an01897e.
Meagher, Robert J., Priye, Aashish, Light, Yooli K., Huang, Cheng, & Wang, Eryu. Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA. United States. doi:10.1039/c7an01897e.
Meagher, Robert J., Priye, Aashish, Light, Yooli K., Huang, Cheng, and Wang, Eryu. Tue . "Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA". United States. doi:10.1039/c7an01897e.
@article{osti_1429722,
title = {Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA},
author = {Meagher, Robert J. and Priye, Aashish and Light, Yooli K. and Huang, Cheng and Wang, Eryu},
abstractNote = {Loop-mediated isothermal amplification (LAMP), coupled with reverse transcription (RT), has become a popular technique for detection of viral RNA due to several desirable characteristics for use in point-of-care or low-resource settings. The large number of primers in LAMP (six per target) leads to an increased likelihood of primer-dimer interactions, and the inner primers in particular are prone to formation of stable hairpin structures due to their length (typically 40-45 bases). Although primer-dimers and hairpin structures are known features to avoid in nucleic acid amplification techniques, there is little quantitative information in literature regarding the impact of these structures on LAMP or RT-LAMP assays. In this study, we examine the impact of primer-dimers and hairpins on previously-published primer sets for dengue virus and yellow fever virus. We demonstrate that minor changes to the primers to eliminate amplifiable primer dimers and hairpins improves the performance of the assays when monitored in real time with intercalating dyes, and when monitoring a fluorescent endpoint using the QUASR technique. We also discuss the thermodynamic implications of these minor changes on the overall stability of amplifiable secondary structures, and we present a single thermodynamic parameter to predict the probability of non-specific amplification associated with LAMP primers.},
doi = {10.1039/c7an01897e},
journal = {Analyst},
number = 8,
volume = 143,
place = {United States},
year = {Tue Mar 27 00:00:00 EDT 2018},
month = {Tue Mar 27 00:00:00 EDT 2018}
}

Journal Article:
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Works referenced in this record:

Clustal W and Clustal X version 2.0
journal, September 2007


MUSCLE: multiple sequence alignment with high accuracy and high throughput
journal, March 2004

  • Edgar, R. C.
  • Nucleic Acids Research, Vol. 32, Issue 5, p. 1792-1797
  • DOI: 10.1093/nar/gkh340