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

Meagher, Robert J.; Priye, Aashish; Light, Yooli K.; Huang, Cheng; Wang, Eryu

doi:10.1039/c7an01897e

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

Journal Article · Tue Mar 27 00:00:00 EDT 2018 · Analyst

DOI:https://doi.org/10.1039/c7an01897e· OSTI ID:1429722

Meagher, Robert J. ^[1]; Priye, Aashish ^[1]; Light, Yooli K. ^[1]; Huang, Cheng ^[2]; Wang, Eryu ^[2]

Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Univ. of Texas Medical Branch, Galveston, TX (United States)

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.

View Accepted Manuscript (DOE)

Cite

Export

Save

Research Organization:: Sandia National Lab. (SNL-NM), Albuquerque, NM (United States)

Sponsoring Organization:: National Institutes of Health (NIH); USDOE

Grant/Contract Number:: AC04-94AL85000

OSTI ID:: 1429722

Report Number(s):: SAND-2017-13686J; ANALAO; 659610

Journal Information:: Analyst, Vol. 143, Issue 8; ISSN 0003-2654

Publisher:: Royal Society of ChemistryCopyright Statement

Country of Publication:: United States

Language:: English

Citation Metrics:

Cited by: 60 works

Citation information provided by
Web of Science

References (31)

A smartphone-based diagnostic platform for rapid detection of Zika, chikungunya, and dengue viruses Priye, Aashish; Bird, Sara W.; Light, Yooli K. Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/srep44778	journal	March 2017
Detection of Loop-Mediated Isothermal Amplification Reaction by Turbidity Derived from Magnesium Pyrophosphate Formation Mori, Yasuyoshi; Nagamine, Kentaro; Tomita, Norihiro Biochemical and Biophysical Research Communications, Vol. 289, Issue 1 https://doi.org/10.1006/bbrc.2001.5921	journal	November 2001
Analysis of genotype diversity and evolution of Dengue virus serotype 2 using complete genomes Waman, Vaishali P.; Kolekar, Pandurang; Ramtirthkar, Mukund R. PeerJ, Vol. 4 https://doi.org/10.7717/peerj.2326	journal	January 2016
Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue Goto, Motoki; Honda, Eiichi; Ogura, Atsuo BioTechniques, Vol. 46, Issue 3 https://doi.org/10.2144/000113072	journal	March 2009
Nearest-Neighbor Thermodynamics and NMR of DNA Sequences with Internal A·A, C·C, G·G, and T·T Mismatches ^† Peyret, Nicolas; Seneviratne, P. Ananda; Allawi, Hatim T. Biochemistry, Vol. 38, Issue 12 https://doi.org/10.1021/bi9825091	journal	March 1999
A real-time reverse transcription loop-mediated isothermal amplification assay for the rapid detection of yellow fever virus Kwallah, Allan ole; Inoue, Shingo; Muigai, Anne W. T. Journal of Virological Methods, Vol. 193, Issue 1 https://doi.org/10.1016/j.jviromet.2013.05.004	journal	October 2013
Surveillance for Western Equine Encephalitis, St. Louis Encephalitis, and West Nile Viruses Using Reverse Transcription Loop-Mediated Isothermal Amplification Wheeler, Sarah S.; Ball, Cameron S.; Langevin, Stanley A. PLOS ONE, Vol. 11, Issue 1 https://doi.org/10.1371/journal.pone.0147962	journal	January 2016
Clustal W and Clustal X version 2.0 Larkin, M. A.; Blackshields, G.; Brown, N. P. Bioinformatics, Vol. 23, Issue 21, p. 2947-2948 https://doi.org/10.1093/bioinformatics/btm404	journal	September 2007
Loop-mediated isothermal amplification of DNA Notomi, T. Nucleic Acids Research, Vol. 28, Issue 12 https://doi.org/10.1093/nar/28.12.e63	journal	June 2000
Detection of Japanese yam mosaic virus by RT-LAMP Fukuta, S.; Iida, T.; Mizukami, Y. Archives of Virology, Vol. 148, Issue 9 https://doi.org/10.1007/s00705-003-0134-5	journal	January 2003
Comparison of fluorescent intercalating dyes for quantitative loop-mediated isothermal amplification (qLAMP) Oscorbin, Igor P.; Belousova, Ekaterina A.; Zakabunin, Aleksandr I. BioTechniques, Vol. 61, Issue 1 https://doi.org/10.2144/000114432	journal	July 2016
Robustness of a loop-mediated isothermal amplification reaction for diagnostic applications Francois, Patrice; Tangomo, Manuela; Hibbs, Jonathan FEMS Immunology & Medical Microbiology, Vol. 62, Issue 1 https://doi.org/10.1111/j.1574-695X.2011.00785.x	journal	June 2011
Loop-Mediated Isothermal Amplification Technology: Towards Point of Care Diagnostics Njiru, Zablon Kithinji PLoS Neglected Tropical Diseases, Vol. 6, Issue 6 https://doi.org/10.1371/journal.pntd.0001572	journal	June 2012
Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products Tomita, Norihiro; Mori, Yasuyoshi; Kanda, Hidetoshi Nature Protocols, Vol. 3, Issue 5 https://doi.org/10.1038/nprot.2008.57	journal	April 2008
Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay Liu, Wei; Huang, Simo; Liu, Ningwei Scientific Reports, Vol. 7, Issue 1 https://doi.org/10.1038/srep40125	journal	January 2017
Quenching of Unincorporated Amplification Signal Reporters in Reverse-Transcription Loop-Mediated Isothermal Amplification Enabling Bright, Single-Step, Closed-Tube, and Multiplexed Detection of RNA Viruses Ball, Cameron S.; Light, Yooli K.; Koh, Chung-Yan Analytical Chemistry, Vol. 88, Issue 7 https://doi.org/10.1021/acs.analchem.5b04054	journal	March 2016
Detection of Acute HIV-1 Infection by RT-LAMP Rudolph, Donna L.; Sullivan, Vickie; Owen, S. Michele PLOS ONE, Vol. 10, Issue 5 https://doi.org/10.1371/journal.pone.0126609	journal	May 2015
A CCD-based fluorescence imaging system for real-time loop-mediated isothermal amplification-based rapid and sensitive detection of waterborne pathogens on microchips Ahmad, Farhan; Seyrig, Gregoire; Tourlousse, Dieter M. Biomedical Microdevices, Vol. 13, Issue 5 https://doi.org/10.1007/s10544-011-9562-2	journal	July 2011
Sequence-specific detection method for reverse transcription, loop-mediated isothermal amplification of HIV-1 Curtis, Kelly A.; Rudolph, Donna L.; Owen, S. Michele Journal of Medical Virology, Vol. 81, Issue 6 https://doi.org/10.1002/jmv.21490	journal	June 2009
Analysis of a Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) for yellow fever diagnostic Nunes, Marcio R. T.; Vianez, João Lídio; Nunes, Keley N. B. Journal of Virological Methods, Vol. 226 https://doi.org/10.1016/j.jviromet.2015.10.003	journal	December 2015
Visual detection of isothermal nucleic acid amplification using pH-sensitive dyes Tanner, Nathan A.; Zhang, Yinhua; Evans, Thomas C. BioTechniques, Vol. 58, Issue 2 https://doi.org/10.2144/000114253	journal	February 2015
A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics SantaLucia, J. Proceedings of the National Academy of Sciences, Vol. 95, Issue 4 https://doi.org/10.1073/pnas.95.4.1460	journal	February 1998
Universal Single-Probe RT-PCR Assay for Diagnosis of Dengue Virus Infections Alm, Erik; Lesko, Birgitta; Lindegren, Gunnel PLoS Neglected Tropical Diseases, Vol. 8, Issue 12 https://doi.org/10.1371/journal.pntd.0003416	journal	December 2014
MUSCLE: multiple sequence alignment with high accuracy and high throughput Edgar, R. C. Nucleic Acids Research, Vol. 32, Issue 5, p. 1792-1797 https://doi.org/10.1093/nar/gkh340	journal	March 2004
Yellow fever virus: Genetic and phenotypic diversity and implications for detection, prevention and therapy Beasley, David W. C.; McAuley, Alexander J.; Bente, Dennis A. Antiviral Research, Vol. 115 https://doi.org/10.1016/j.antiviral.2014.12.010	journal	March 2015
Robust Strand Exchange Reactions for the Sequence-Specific, Real-Time Detection of Nucleic Acid Amplicons Jiang, Yu Sherry; Bhadra, Sanchita; Li, Bingling Analytical Chemistry, Vol. 87, Issue 6 https://doi.org/10.1021/ac504387c	journal	March 2015
The effect of main urine inhibitors on the activity of different DNA polymerases in loop-mediated isothermal amplification Jevtuševskaja, Jekaterina; Krõlov, Katrin; Tulp, Indrek Expert Review of Molecular Diagnostics, Vol. 17, Issue 4 https://doi.org/10.1080/14737159.2017.1283218	journal	January 2017
Colorimetric Detection of Dengue by Single Tube Reverse-Transcription-Loop-Mediated Isothermal Amplification Lau, Yee-Ling; Lai, Meng-Yee; Teoh, Boon-Teong PLOS ONE, Vol. 10, Issue 9 https://doi.org/10.1371/journal.pone.0138694	journal	September 2015
Development of a TaqMan Array Card for Acute-Febrile-Illness Outbreak Investigation and Surveillance of Emerging Pathogens, Including Ebola Virus Liu, Jie; Ochieng, Caroline; Wiersma, Steve Journal of Clinical Microbiology, Vol. 54, Issue 1 https://doi.org/10.1128/JCM.02257-15	journal	October 2015
Simultaneous multiple target detection in real-time loop-mediated isothermal amplification Tanner, Nathan A.; Zhang, Yinhua; Evans, Thomas C. BioTechniques, Vol. 53, Issue 2 https://doi.org/10.2144/0000113902	journal	August 2012
Complete Genome Sequence of Clostridium botulinum CJ0611A1, a Type A(B) Isolate Associated with an International Outbreak of Botulism from Commercial Carrot Juice Harris, Richard; Dussault, Forest; Flint, Annika Microbiology Resource Announcements, Vol. 10, Issue 1 https://doi.org/10.1128/mra.01111-20	journal	January 2021

Cited By (4)

Rapid sex determination of a wild passerine species using loop‐mediated isothermal amplification (LAMP) Koch, Hanna R.; Blohm‐Sievers, Elke; Liedvogel, Miriam Ecology and Evolution, Vol. 9, Issue 10 https://doi.org/10.1002/ece3.5168	journal	April 2019
A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection Zhou, Wenhua; Hu, Li; Ying, Liming Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-07324-5	journal	November 2018
Polymerase chain reaction combined with fluorescent lateral flow immunoassay based on magnetic purification for rapid detection of canine parvovirus 2 Zhuang, Linlin; Ji, Yongxin; Tian, Peilong BMC Veterinary Research, Vol. 15, Issue 1 https://doi.org/10.1186/s12917-019-1774-3	journal	January 2019
Field diagnosis and genotyping of chikungunya virus using a dried reverse transcription loop-mediated isothermal amplification (LAMP) assay and MinION sequencing Hayashida, Kyoko; Orba, Yasuko; Sequeira, Patricia C. PLOS Neglected Tropical Diseases, Vol. 13, Issue 6 https://doi.org/10.1371/journal.pntd.0007480	journal	June 2019