Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA
| العنوان: | Impact of primer dimers and self-amplifying hairpins on reverse transcription loop-mediated isothermal amplification detection of viral RNA |
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| المؤلفون: | Aashish Priye, Eryu Wang, Robert J. Meagher, Yooli Kim Light, Cheng Huang |
| المصدر: | The Analyst. 143:1924-1933 |
| بيانات النشر: | Royal Society of Chemistry (RSC), 2018. |
| سنة النشر: | 2018 |
| مصطلحات موضوعية: | 0301 basic medicine, Loop-mediated isothermal amplification, Dengue virus, medicine.disease_cause, Sensitivity and Specificity, 01 natural sciences, Biochemistry, Article, Virus, Analytical Chemistry, 03 medical and health sciences, Primer dimer, Electrochemistry, medicine, Environmental Chemistry, Reverse Transcription Loop-mediated Isothermal Amplification, Spectroscopy, DNA Primers, Base Sequence, Chemistry, 010401 analytical chemistry, Reverse Transcription, Nucleic acid amplification technique, Dengue Virus, Molecular biology, Reverse transcriptase, 0104 chemical sciences, 030104 developmental biology, RNA, Viral, sense organs, Primer (molecular biology), Nucleic Acid Amplification Techniques |
| الوصف: | 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 that can be correlated to the probability of non-specific amplification associated with LAMP primers. |
| تدمد: | 1364-5528 0003-2654 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::5866e87dd7834b873858e26937a04010 https://doi.org/10.1039/c7an01897e |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....5866e87dd7834b873858e26937a04010 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 13645528 00032654 |
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