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 that can be correlated to the probability of non-specific amplification associated with LAMP primers.

A CRISPR–Cas9-triggered strand displacement amplification method for ultrasensitive DNA detection

“…It is noticed that as the concentration of pTF1 target is lowered, the intensity corresponding to CRISDA-specific product decreases, while non-specific products with lower molecular weight derived from primer dimers increase. This phenomenon has been observed in various nucleic acid amplification techniques such as traditional PCR reactions, HAD, and LAMP 6 , 32 , 33 . Hence, to quantitatively distinguish the target-specific product from non-target-specific products, a PNA invasion-mediated endpoint measurement is performed by incubating 20 μL of the amplification products with a biotin-labeled PNA and a Cy5-labeled PNA probe (both at 100 nM) targeting the middle region of the amplicon, which is subject to magnetic pull-down and fluorescence measurements (Fig.…”

Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

“…With each amplification, and re-prime by FIP, a single product is generated. This process of hairpin accumulation would cause the linear 'rinsing' baseline observed by other researchers (37).…”

“…Thus, accumulation of a sufficient pool of randomers may take time, but still result in a delayed bulk exponential amplification event. Furthermore, should a hairpin with attached randomer form, it is possible that the rising baseline, attributed to hairpin formation (37), may also by in vitro selection of the products, lead to and result in spontaneous exponential amplification.…”

Rapid sex determination of a wild passerine species using loop‐mediated isothermal amplification (LAMP)

“…Consequently, one of our goals was to find one single incubation temperature that would work accurately for both CHD-W and CHD-Z primer sets so that separate reactions could be run simultaneously on the same thermoblock, requiring less equipment and time overall. False-positive results may also be obtained through primer-dimer formation, which is more likely to occur when high concentrations of primers are used, as are in a LAMP assay (Meagher, Priye, Light, Huang, & Wang, 2018 (Ball et al, 2016;Hsieh et al, 2014;Wang, Brewster, Paul, & Tomasula, 2015). Therefore, The most commonly reported drawback of LAMP is high rates of false positives.…”

“…High temperature, humidity, and inadequate volume of reagents are known risk factors contributing to carry over contamination (Nagai et al, 2016). False-positive results may also be obtained through primer-dimer formation, which is more likely to occur when high concentrations of primers are used, as are in a LAMP assay (Meagher, Priye, Light, Huang, & Wang, 2018). Herein, false positives occurred in the template-free controls during primer optimization.…”