Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
17 04 2020
17 04 2020
Historique:
accepted:
06
02
2020
revised:
08
01
2020
received:
04
10
2019
pubmed:
28
2
2020
medline:
7
7
2020
entrez:
28
2
2020
Statut:
ppublish
Résumé
Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.
Identifiants
pubmed: 32103255
pii: 5760753
doi: 10.1093/nar/gkaa099
pmc: PMC7144905
doi:
Substances chimiques
DNA
9007-49-2
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
e42Subventions
Organisme : NIGMS NIH HHS
ID : T32 GM112592
Pays : United States
Informations de copyright
© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.
Références
Analyst. 2018 Apr 16;143(8):1924-1933
pubmed: 29620773
Nano Lett. 2006 Mar;6(3):520-3
pubmed: 16522055
Anal Chem. 2019 Oct 15;91(20):12784-12792
pubmed: 31525952
Sci Rep. 2017 Jun 26;7(1):4253
pubmed: 28652587
Sci Rep. 2017 Jan 06;7:40125
pubmed: 28059137
Biol Chem. 2007 Apr;388(4):367-72
pubmed: 17391057
Anal Chem. 2017 Nov 7;89(21):11529-11536
pubmed: 29027789
J Microbiol Methods. 2017 Jan;132:41-45
pubmed: 27984058
Taiwan J Obstet Gynecol. 2011 Sep;50(3):261-7
pubmed: 22030037
Nucleic Acids Res. 2000 Jun 15;28(12):E63
pubmed: 10871386
J Virol Methods. 2011 Apr;173(1):67-74
pubmed: 21256868
Anal Chem. 2018 Jun 5;90(11):6580-6586
pubmed: 29667809
Biotechniques. 2001 Nov;31(5):1122-4, 1126, 1128-9
pubmed: 11730018
ACS Nano. 2017 Mar 28;11(3):2934-2943
pubmed: 28234452
Sci Rep. 2017 Mar 28;7:45601
pubmed: 28349967
PLoS One. 2016 Oct 19;11(10):e0163060
pubmed: 27760148
Biotechniques. 2001 Apr;30(4):852-6, 858, 860 passim
pubmed: 11314268
Nucleic Acids Res. 2016 Jan 29;44(2):e10
pubmed: 26358811
Electrophoresis. 2019 Oct;40(20):2706-2717
pubmed: 31206723
PLoS Negl Trop Dis. 2018 Dec 20;12(12):e0006900
pubmed: 30571705
J Med Microbiol. 2008 Jan;57(Pt 1):50-57
pubmed: 18065667
Sci Adv. 2018 Sep 28;4(9):eaat6459
pubmed: 30263958
Anal Chem. 2012 Oct 2;84(19):8371-7
pubmed: 22947054
Biotechniques. 2012 Aug;53(2):81-9
pubmed: 23030060
Sci Rep. 2017 Oct 24;7(1):13928
pubmed: 29066799
Sci Rep. 2018 Apr 3;8(1):5548
pubmed: 29615801
Nat Biotechnol. 2010 Jun;28(6):595-9
pubmed: 20495550
Mol Cell Probes. 2010 Aug;24(4):190-5
pubmed: 20230890
Anal Chem. 2013 Nov 19;85(22):11129-36
pubmed: 24199852
Anal Chem. 2019 Sep 3;91(17):11275-11282
pubmed: 31356737
PLoS One. 2015 Apr 09;10(4):e0123126
pubmed: 25856093
J Mol Biol. 2004 May 28;339(2):395-404
pubmed: 15136041
Anal Chem. 2013 Feb 5;85(3):1540-6
pubmed: 23324061
Sci Rep. 2017 Nov 6;7(1):14586
pubmed: 29109452
Anal Chem. 2015 Mar 17;87(6):3314-20
pubmed: 25708458
PLoS Negl Trop Dis. 2008 Feb 06;2(1):e147
pubmed: 18253475
Biochemistry. 2004 Dec 28;43(51):16515-24
pubmed: 15610046
Sci Rep. 2017 Feb 08;7:42326
pubmed: 28176860
Anal Chem. 2016 Apr 5;88(7):3562-8
pubmed: 26980448
J Pathog. 2014;2014:183167
pubmed: 25614838
Anal Chem. 2019 Jan 2;91(1):1034-1042
pubmed: 30565936