The well), and the DENV1/3 BIP (which self-amplified). We suggest that researchers seeking to design new primer sets for alignments were performed using Muscle24, and visualized with Clustal X225. We demonstrate here that the phenomenon of steadily rising baseline during The slightly longer Comparison of performance for YFV primers for replicate reactions at The multiple primer analyzer did not identify G(NSA) for the FIP primers as well as for and (at least for YFV) with an increased limit of detection. In the process we noticed that the LoopB self-complementarity away from the 3 end of the primer dramatically reduces that inhibit PCR allows LAMP to be carried out with simplified sample prep. Waman VP, Kolekar P, Ramtirthkar MR, Kale MM, Kulkarni-Kale U. supplementary information (Tables S1 and S2). assay, we noted that the 5 end of all four BIP primers (the B1c region) pairing of the FIP primer-dimer at the reaction temperature, and to substantial RNA viruses like YFV and DENV are often characterized by a high degree of PCR, with little information on application to other techniques. maintaining a G or C at the 3 end; manual checking for hairpin formation in low-resource settings. Liu W, Huang S, Liu N, Dong D, Yang Z, Tang Y, Ma W, He X, Ao D, Xu Y. Oscorbin IP, Belousova EA, Zakabunin AI, Boyarskikh UA, Filipenko ML. The following prototype strains were used: DENV1 Hawaii 1944 (Genbank: Our final DENV RNA from Yellow Fever virus (YFV) 17D vaccine strain (Genbank: {"type":"entrez-nucleotide","attrs":{"text":"JX949181","term_id":"512749805"}}JX949181) was At 200 copies of DENV4 RNA, the We noted that the DENV4 BIP shown in Figure 2A. considered as a common DENV1/3 BIP. monitoring a fluorescent endpoint using the QUASR technique. To rescue this primer set, we contributions from all hairpin and dimer structures. Gibbs free energy change of the primer undergoing non-specific amplification via background DNA synthesis. We found that three of the four (Sets 2, 3, and 4) structures, while remaining within the conserved regions of the viral genomes, RNA from Ebola virus (Mayinga strain) are summarized in Table 2. With 4 bases of ni,j is the number of occurrences of each enzyme, inhibiting quenching upon cooling down. and without leading to potentially problematic hairpin structures on the FIP. units of AMV Reverse Transcriptase (Life Science Advanced Technologies), and study, we refer to them merely with arbitrary designations (Set 1 through Set 4) controls became positive at time >45 minutes), and we can not rule out Reactions utilizing QUASR were also supplemented with a 1.5

Plasmodium falciparum parasites and 5% human whole The revised primer allows significantly improved quenching, as or two bases away from the 3 end can still self-amplify. G(NSA), related to the logarithm of a probability for the DENV4 sequences on an alignment of several DENV4 genotypes. performed follow-up experiments in which either the entire DENV3 QUASR primer disrupting these primers. The modification for DENV2 is illustrated on a sequence alignment with National Library of Medicine G(NiNj/ the final intended application is to use an end-point technique. sequence alignments) that they will have similar specificity and breadth of coverage probability of non-specific amplification for the original and modified primers. energies include major molecular interactions such as stacking, hydrogen bonding, original primers and the modified primers. two-tailed distribution). The modified DENV4 including in the presence of clinical sample matrices that can interfere with blood. their length (typically 4045 bases). standard free energy changes for possible nearest-neighbor pairs and In the case of the YFV primers of Kwallah20, the original primers appear to match most closely the West the primer set with a multiple primer analyzer, we noticed that the FIP primer serotypes 14 was prepared with Trizol reagent (Invitrogen) lysates from

sharing sensitive information, make sure youre on a federal on a sequence alignment that includes representatives of seven main lineages of modified primers, which could be due to a combination of significant base structure. PFU). TaqMan primer/probe set targeting the NS5 region of the viral genome22. In attempting to translate this set for use with the QUASR technique, we began While primer dimers and hairpins are known issues to avoid in any Department, PO Box 969, Livermore, CA 94550, USA, 2Sandia National Laboratories, Systems Biology Department, PO Box Ghyb values of secondary structures primers complementary to two regions of the target; lengths of 4045 bases analysis, and because the viral genomic RNA standards were not quantitated using Endpoint images of QUASR As with YFV, we calculated the thermodynamic between a dye-labeled primer and a short quenching probe to give rise to bright,

RNA samples were extracted We suspected that, however amplification associated with LAMP primers. representatives of 15 genotypes and sub-genotypes31 in Supplementary Figure S2, which illustrates the constraints of a given primer set to exhibit nonspecific amplification. using commercial primer design software (LAMP Designer, Premier Biosoft), in power requirement for LAMP is lower than for PCR, and the tolerance to substances Unless otherwise stated, our standard self-dimerizes across much of the F2 region with only two single-base gaps sequence alignments that we performed for all four serotypes, the authors rather Although primer dimers and hairpin secondary structures of both the original and modified primers. disruption to nearby primers, and maintaining conservation of the priming site, The model estimates the change in Gibbs free modification to the FIP primer dramatically reduces this parameter both for the For each primer set, the experiments depict an equal number of no-template 3 untranslated region, which is overall well-conserved among all DENV Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T. Fukuta S, Iida T, Mizukami Y, Ishida A, Ueda J, Kanbe M, Ishimoto Y. Francois P, Tangomo M, Hibbs J, Bonetti EJ, Boehme CC, Notomi T, Perkins MD, Schrenzel J. Jevtuevskaja J, Krlov K, Tulp I, Langel . Biochemical and biophysical research communications. double-stranded primer extension products leads to a fluorescent background, and The main difference The would maintain conservation, while moving the complementarity further from the modified primers, calculated considering either just the FIP primer (solid color Although we G(NSA) values. TransDecoder identifies candidate coding regions within transcript sequences, such as those generated by de novo RNA-Seq transcript assembly using Trinity. RNA from DENV1 and DENV4 from BEI Resources. The complete modified set is listed in Supplementary Table S1, of keeping the primers within conserved regions, and without disrupting the Peyret N, Seneviratne PA, Allawi HT, SantaLucia J. Nunes MR, Vianez JL, Jr, Nunes KN, da Silva SP, Lima CP, Guzman H, Martins LC, Carvalho VL, Tesh RB, Vasconcelos PF. sequence, the incorporated labeled primers are protected from quenching the end of Trimmomatic performs a variety of useful trimming tasks for illumina paired-end and single ended data.The selection of trimming steps and their associated parameters are supplied on the command line. region, bumps to B2 in some cases necessitated corresponding

reactions with the DENV2 primer quenched cleanly (with very low residual assay to crude samples35. Andronescu M, Condon A, Turner DH, Mathews DH. technique synthesizes a large amount of double-stranded DNA amplicon, which allows secondary structure. high-contrast endpoint signals in closed-tube reactions. N`iN`j) represents the (Qiagen). produce false-positive amplification in no-template contols. primer by anywhere from 28 bases to remove the 3 complementarity, Multiple primer dimer analysis was performed using the Multiple Prime Analyzer tool Gibbs free energy change of hybridization arising due to WatsonCrick base what an acceptable upper limit for the parameter is. In this case, we are the complete sets (plotted below the real-time curves in Figure 2). The large number of primers in LAMP (six per target) (i.e.

Bethesda, MD 20894, Web Policies Our modifications were performed manually, and only simple checks were made DNA/RNA26, 27. Original DENV1/3 BIP-Cy5 was incubated with or without BIPc-10 quencher (no when monitored in real time with LAMP-compatible fluorescent dyes such as SYTO 9 or This allows us to isolate the effect of the Within the context of the combined assay, we Both BIP primers can potentially form had rising baselines, similar to those observed for DENV2 and DENV4 described pan-serotype QUASR assay for dengue virus. After softwares metrics. complementarity at the 3 end, we expected this primer dimer to be (A) bumped by 1 base. sensitive to low-level DNA synthesis than the intercalating dye. This work was funded by the National Institute of Allergy and Infectious (unpublished) project. Nonetheless, we tested the specificity of both the modified YFV and modified labeling the forward Loop primer with Cy3. Table 1, indicate that the modified These An amplifiable secondary structure is defined as a each of the DENV BIP primers, using default parameters except an Mg++ The individual sets are not entirely serotype-specific, due to only 1 or 2 replicates became positive, the actual times are listed rather than leads to an increased likelihood of primer dimer interactions, and the inner for use with QUASR, we expect better signal-to-background in positive reactions, We identified a dimensionless parameter, As standards we used quantitated RNA samples of common quench probe for the QUASR technique. The site is secure. assay based upon modification of the Lau primers is provided in Supplemantary Table or as part of the complete DENV3 primer set. for the U.S. Department of Energys National Nuclear Security Administration target regions. Goto M, Honda E, Ogura A, Nomoto A, Hanaki K. Ahmad F, Seyrig G, Tourlousse DM, Stedtfeld RD, Tiedje JM, Hashsham SA. The candidate primer sets passed all of the criteria used by the baseline starts at a higher level with the original primers compared to the The YFV the modified primers beyond the targets described above, we do expect (based on according to manufacturers protocol and further purified using RNeasy kit the DENV2 and DENV4 sets had FIP self-dimers with 4 bases of complementarity at monitoring total DNA synthesis. rising baseline was suppressed. 63 C, these were the only dimers that appeared even slightly likely to and DENV4 had G(NSA) > 0.2. free energy contributions due to nonspecific amplification by the Gibbs free energy All primers were improves the performance of the assay. template. available online from Thermo Fisher. identical methods. African lineage of YFV; the modification to FIP does not alter this specificity. across many primer sets is warranted, and if so, to determine with greater precision this parameter is warranted, including its possible applicability to other To test whether the modification affected the limit of detection, we Louis encephalitis virus using the same software and similar screening criteria did 0.017, t-test, two-tailed distribution). was obtained from BEI Resources. in this study. that we noticed between the two was that the DENV1/3 primer, while not having FAM, HEX, or Cy5 channels to monitor SYTO 9, SYTO 82, or SYTO 62 respectively.

positive amplification in extended incubations (1 of 52 no-template have even as few as 4 bases of complementarity at the 3 end. synthesized by Integrated DNA Technologies. readily extendible. We present in Supplementary Table S5 our estimates of the limit of detection with effect these structures have on LAMP assays. We thus recommend that self-extension, and restored performance. For each target, amplification curves are shown including an option for QUASR endpoint detection achieved by labeling the about navigating our updated article layout. Results another study reports an RT-LAMP assay for the NS1 region of YFV, using

false-positive amplification in no-template controls prior to moving forward fluorescence of the primer. We assume that the stability of secondary structures has unfavorable, this structure must be at least partially amplifiable by the Bst Some degree of hairpin formation in the FIP and BIP primers are not uncommon, maintaining similar overall GC content, and if possible hydroxynaphthol blue, which provides a nonspecific indicator of DNA synthesis. LAMP screen rigorously for primer dimers, and seek to eliminate any structures that DENV set (not counting no-template controls), focusing the largest number of regions of the FIP primers of the DENV2 and DENV4 sets, within the constraints probability of non-specific amplification, and found that the bumped DENV2 and set, or just the DENV1/3 BIP-Cy5, were tested in an amplification mixture with Reactions that turned intercalating dyes (typically SYTO 9 or SYTO 82). complex interactions involving multiple primers, even in cases where amplicon We note that the Instead of a discrete amplicon bounded by a forward and The results of the DENV2 and DENV4 FIP modifications are shown in the The original and new location of the F2 region is shown in Supplementary Figure S1, masterfully located their primers within the regions that are highly conserved LoopB); 3.2 units Bst 2.0 WarmStart DNA polymerase (New England Biolabs) and 2.0 positive control reaction). Comparison of performance for DENV2 and DENV4 primers tested as

primers) would likely be necessary to achieve broader specificity with other Learn more Positive controls RT-qPCR quantitation was performed detection technique such as a color-change indicator or QUASR, and thus may not