A common problem in nucleic acid amplification reactions is undesired activity from DNA polymerases during room temperature reaction setup (26, 27). This activity may not be a problem when performing a limited number of samples set up by hand, but it can result in irreproducibility for medium- or high-throughput or automated workflows. To test whether LAMP and DARQ detection may be affected by preincubation at room temperature, identical LAMP reactions were set up and performed with or without a 2-h preincubation at 25°C. Both Bst DNA polymerase large fragment and Bst 2.0 DNA polymerase were negatively impacted by the 2-h preincubation, likely due to low levels of DNA polymerase activity at 25°C (Figure 2). In PCRs, this problem is typically overcome through the use of “hot start” DNA polymerases. Bst 2.0 WarmStart DNA polymerase provides this functionality for isothermal amplification techniques through an engineered aptamer that inhibits polymerase activity below 50°C (28), and its efficacy in DARQ is shown in Figure 2. Bst 2.0 WarmStart maintains identical amplification profiles whether LAMP is performed immediately after set up (Figure 2A) or after 2 h preincubation at 25°C (Figure 2B). This benefit accommodates room temperature reaction setup, but otherwise does not affect Bst 2.0 performance, so all subsequent data are from standard Bst 2.0.
With DARQ detection validated, we next sought to extend the method to multiple targets detected in a single reaction. Figure 3 presents fluorescence curves from LAMP reactions containing two distinct, complete primer sets and their corresponding genomic DNA targets. For multiple target amplification, we maintained the total amount of primer in a standard LAMP reaction; using each set at full concentration resulted in suboptimal performance as concentration increased (i.e., total primer concentration was kept to 4.4 μM regardless of the number of templates, with each primer set adjusted by 1/n, where n is number of targets in the reaction). As shown in Figure 3, DARQ robustly detects distinct targets in a single LAMP reaction. Curves shown are normalized to maximum fluorescence signal in that channel to account for differences in the quantum yield of various fluorophores. The detection provided robust signal for each target regardless of the speed of their independent amplification, which will vary based on the nature of the primers, templates, and target copy number. Despite the quicker amplification reaching exponential phase sooner, the slower amplification was observed to proceed unaffected, obviating the need for consideration of amplification speed when multiplex targets are selected (Figure 3B). This independent nature of DARQ reactions allows maintenance of sensitivity when performed with multiple targets, as shown in Figure 3C, where FAM-CFTR is detected to ~2.9 copies of HeLa genomic DNA (10 pg) in the same reaction as robust LAMP for ROX-lec-10 (82.5 ng C. elegans DNA, ~7.6 × 105 copies). Thus a robust LAMP standard curve can be generated across a copy number range of Target 1 (here, CFTR) while Target 2 (lec-10) is detected simultaneously. Amplification of the constant target remains unchanged (all 5 ROX Ct values 11.8 ± 0.03 min) across the copy number range of the variable target providing a reliable positive control (Figure 3C). This property allows LAMP to be performed with an internal standard, an important consideration for diagnostic applications. Figure 3C demonstrates DARQ performance at low copy numbers, but high copy numbers are also reliably detected, as seen in Figure 2 (5 ng E. coli genomic DNA, ~106 copies) and Figure 3A (5 ng λ DNA, ~108 copies). Use of DARQ detection thus imposes no limitation to the sensitivity of the LAMP reaction. Similarly, the dynamic range of LAMP is unaffected by duplex DARQ, which maintains robust detection from 10–108 copies.
DARQ can easily be extended to three and four target reactions (Figure 4), again with total primer concentration constant and each set adjusted for number of targets. Reducing primer concentration 3- or 4-fold does accordingly increase time to reach threshold. This drop in time was consistent, making template quantification reliable, and the reaction times were still rapid with Bst 2.0 DNA polymerase. The multiplexed reactions display robust amplification of three (Figure 4A) or four (Figure 4B) targets, with loss of signal amplitude accompanying decreased concentration of the fluorophore-containing primer. This fluorescence decrease emphasizes the need for bright fluorophores, with high quantum yield and appropriate spectral matching with the fluorescence detection channels. In DARQ reactions, ROX gave the best results (>20,000 background-subtracted fluorescence counts in single-plex reactions), likely due to a 15 nm wider detection channel. Cy5, Cy5.5, and HEX gave similarly high signal (10,000–15,000), but 6-FAM (<5,000) gave lower fluorescence signal that became difficult to distinguish from background fluorescence when diluted 3- or 4-fold for multiplex reactions. Thus DARQ is accommodating of any fluorophore that can be quenched and detected, but for detecting more than three targets simultaneously brighter dyes are preferred.