Lack of correlation between reaction speed and analytical sensitivity in isothermal amplification reveals the value of digital methods for optimization: validation using digital real-time RT-LAMP

Abstract

In this paper, we asked if it is possible to identify the best primers and reaction conditions based on improvements in reaction speed when optimizing isothermal reactions. We used digital single-molecule, real-time analyses of both speed and efficiency of isothermal amplification reactions, which revealed that improvements in the speed of isothermal amplification reactions did not always correlate with improvements in digital efficiency (the fraction of molecules that amplify) or with analytical sensitivity. However, we observed that the speeds of amplification for single-molecule (in a digital device) and multi-molecule (e.g. in a PCR well plate) formats always correlated for the same conditions. Also, digital efficiency correlated with the analytical sensitivity of the same reaction performed in a multi-molecule format. Our finding was supported experimentally with examples of primer design, the use or exclusion of loop primers in different combinations, and the use of different enzyme mixtures in one-step reverse-transcription loop-mediated amplification (RT-LAMP). Our results show that measuring the digital efficiency of amplification of single-template molecules allows quick, reliable comparisons of the analytical sensitivity of reactions under any two tested conditions, independent of the speeds of the isothermal amplification reactions.

Development of Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) Assays Targeting SARS-CoV-2

“…In fact, average threshold time of "S_1-2-2" and "N_21" primer sets for 1000 copies were faster than that of "Nsp3_1-61": 7.33 (RPR: 2/3) and 5.06 (RPR: 1/3) minutes, respectively. This dis-relation is previously reported [24].One peculiar observation is that both "S_1-2-2" and "N_21" showed good sensitivity to cDNA template ( Supplementary Figure 1) unlike to RNA. Observed difference of sensitivity to RNA and cDNA seems significant even we account slight amplification that might happened during reverse transcription.…”

“…Observed difference of sensitivity to RNA and cDNA seems significant even we account slight amplification that might happened during reverse transcription. The reason may include stochastic nature of forming proper LAMP amplification intermediate -"dumbbell" structure [24], higher stability of DNA:RNA double strand than DNA:DNA double strand and more.…”

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

“…We found that TTP can be heterogeneous while T m is constant (28.6 ± 8.9 min with 87.5 ± 0.2 • C), indicating that the same product may initiate at different times ( Figure 5F). This is consistent with our knowledge of the stochastic initiation of LAMP (23,(38)(39). Further, we observed some variability in the maximum rate despite similar T m (23.7 ± 6.8 RFU/30 s, with 87.5 ± 0.2 • C T m ), which indicates the same product may amplify at different velocities ( Figure 5G).…”

Digital droplet LAMP as a microfluidic app on standard laboratory devices

“…During assay optimization, the concentration of primers is usually varied to nd the optimum value. As recently reported, 27 the primer concentrations optimized in bulk reactions cannot necessarily be transferred to a small-volume digital LAMP assay. Too few primers might lead to a low efficiency of ddLAMP, as droplets might contain not enough primers to ensure reliable and fast amplication.…”