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Simultaneous multiple target detection in real-time loop-mediated isothermal amplification
 
Nathan A. Tanner, Yinhua Zhang, and Thomas C. Evans
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Supplementary Material

Materials and methods

LAMP primers were designed either manually or using PrimerExplorer V4 (Eiken Chemical). Sequences can be found in Supplementary Table S1, and all synthetic oligonucleotide primers, Q-FIP, and Fd were synthesized by Integrated DNA Technologies (Coralville, IA, USA). The dark quencher was either Iowa Black FQ or RQ, and fluorophores used were 6-FAM, HEX, ROX, Cy5, and Cy5.5, each corresponding to one of five channels in a CFX96 Real Time System (Bio-Rad Laboratories, Hercules, CA, USA), used for performing LAMP reactions (Supplementary Table S2).

Q-FIP:Fd duplexes were annealed by heating 50 μM Q-FIP and 50 μM Fd to 98°C and slowly cooling the mixture to room temperature. LAMP reactions with Bst 2.0 DNA polymerase or Bst 2.0 WarmStart DNA polymerase (New England Biolabs, Ipswich, MA, USA) were performed in 1× Isothermal Amplification Buffer (New England Biolabs): 20 mM Tris-HCl (pH 8.8, 25°C), 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween-20 supplemented to 8 mM MgSO4, and 1.4 mM each of dATP, dCTP, dGTP, and dTTP. LAMP reactions with standard Bst DNA polymerase, LF (New England Biolabs) used a similar buffer based on ThermoPol DF (New England Biolabs), identical in composition as above but with 10 mM KCl in place of 50 mM KCl. LAMP reactions contained: 1.6 μM FIP (or 0.8 μM FIP and 0.8 μM Q-FIP:Fd), 1.6 μM BIP, 0.2 μM F3 and B3, 0.4 μM LoopF and LoopB, and 0.64 U/μL Bst DNA polymerase, LF, Bst 2.0 DNA polymerase, or Bst 2.0 WarmStart DNA polymerase. For multiplex reactions, total primer concentrations were kept to those described for the standard LAMP reaction, but with each set representing 1/n of the total, where n is the number of targets. Bacteriophage λ genomic DNA (5 ng/reaction) and HeLa genomic DNA (100 ng/reaction) were from New England Biolabs, Escherichia coli genomic DNA (5 ng/reaction) was from Affymetrix (Santa Clara, CA, USA), and Caenorhabditis elegans genomic DNA (82.5 ng/reaction) was purified using standard procedures. Reactions were performed at 65°C in triplicate, and all presented Ct values represent an average ± standard deviation.

Results and discussion

In DARQ, the standard FIP primer is modified with a 5′ quencher and annealed to an F1c-complementary detection probe, Fd (Figure 1). To test this method for LAMP detection, we designed four sets of LAMP primers with Q-FIP and accompanying Fd probes, each with a different fluorophore and quencher pair. These targeted genes from different organisms and genome complexities: E. coli dnaE (Iowa Black RQ/Cy5); C. elegans lec-10 (RQ/ROX); human cystic fibrosis transmembrane conductance regulator (CFTR; FQ/6-FAM); and human BRCA1 (RQ/Cy5.5). Additionally, we adapted a set of LAMP primers for bacteriophage λ DNA (5) with the quencher and fluorophore positions reversed (5′-HEX FIP/3′-FQ) to examine any effect of quencher/fluorophore location. Q-FIP:Fd duplexes were made for each primer set, and LAMP reactions performed using duplex FIP primers.

Figure 2 shows LAMP amplification of dnaE from E. coli genomic DNA with DARQ detection using Bst DNA polymerase, large fragment or standard and WarmStart versions of Bst 2.0 DNA polymerase. Release of quenching can be seen as an increase in Cy5 signal, and use of all three polymerases resulted in robust amplification and signal (Figure 2A). The reaction was found to proceed faster when using either the standard or WarmStart versions of Bst 2.0 DNA polymerase as compared with wild-type Bst DNA polymerase (Figure 2 and Supplementary Figure S1). Therefore, Bst 2.0 DNA polymerase was used in all subsequent experimentation. Use of 5′ modified FIP primer alone had no effect on LAMP, but we observed some relative inhibition of amplification when Q-FIP:Fd duplex primer completely replaced the standard FIP primer (Supplementary Figure S1). This inhibition was significantly reduced through the use of equimolar standard FIP primer and Q-FIP:Fd duplex (Supplementary Figure S2). This effect was likely due to faster target generation with FIP and easier incorporation of duplex FIP during exponential amplification, and using equimolar amounts maintains rapid threshold detection with high fluorescence signal amplitude, important for detecting amplification in multiplex reactions.




Figure 2.  DARQ detection in real-time LAMP reactions. (Click to enlarge)


A convenient feature of LAMP F1c regions is that they are typically 20–25 bases long and are selected by Primer Explorer to feature Tm greater than 60°C. Thus by the nature of the LAMP primer design, the F1c:Fd duplex remains stably annealed in our reactions at 63°–65°C, and no signal is observed in the absence of strand-displacing DNA polymerase. However, if nonstandard primer sequences are required, DARQ reactions can simply be performed at lower temperatures to accommodate less stable duplexes. The F1c regions described here range in Tm from 61°–74°C (Supplementary Table S1), and all perform DARQ LAMP reactions at 60°–65°C, indicating that use of F1c:Fd duplexes does not limit primer design considerations. As described above, we also tested a primer pair with fluorophore and quencher positions switched on FIP and Fd. Use of this reverse orientation primer set (λ) resulted in no difference in amplification detection efficiency (Figure 3A and Figure 4) or duplex primer inhibition levels (data not shown), thus we can conclude that DARQ primers can be synthesized with either a 5′ quencher or fluorophore FIP if necessary to accommodate limited modified oligonucleotide synthesis chemistry.

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