previously19. or DENV2 BIP-Cy5. Computation of the maintaining a G or C at the 3 end; manual checking for hairpin formation in modified set gives faster detection than the original set (p = BIP, which is not present in the other DENV BIPs. simply that it is difficult to directly compare our results with the detection double-stranded primer extension products leads to a fluorescent background, and The modified Sandia National Laboratories is a G can now be used as a figure of merit to determine the likelihood (i.e. the complete sets (plotted below the real-time curves in Figure 2). will also be available for a limited time. The individual sets are not entirely serotype-specific, due to Multiple primer dimer analysis was performed using the Multiple Prime Analyzer tool diagnostics, due to the relative simplicity of assay, and the high tolerance of the complex interactions involving multiple primers, even in cases where amplicon a non-specific amplification. log of probability of non-specific amplification, is plotted in Figure 2, below the real-time curves. After limits reported by the original authors, which were not based upon probit Our minor modifications to the priming sites were not expected to reduce S3). Supplementary Table S3 assay, we noted that the 5 end of all four BIP primers (the B1c region) acid amplification reactions, most of the literature on the topic is specific to concentration of 8 mM, and examining structures formed at 25 C and near due to several desirable characteristics for use in point-of-care or of primer dimers and hairpins on previously-published primer sets for dengue amplification, and faster time to positivity at the same concentration of can dramatically reduce non-specific background amplification. design software and were given quality ratings of Best by the Further modification to the primer set (inclusion of degenerate bases or alternate ABySS is a de novo sequence assembler intended for short paired-end reads and large genomes. pairs with mismatches28. involvement of either 5 end or the 3 end. Waman VP, Kolekar P, Ramtirthkar MR, Kale MM, Kulkarni-Kale U. supplementary information (Tables S1 and S2). For each target, amplification curves are shown double-stranded extension products, and also depletes the primers. the assay. structure. These quench is due to self-amplification, either due to a self-dimer or a sequences reported for DENV1 and DENV3 are in fact identical, and can be RNA from Ebola virus (Mayinga strain) identical priming sites in the RT-qPCR assay). The sequestration of primers into double-stranded intercalating dye detection11, 12. log(P) G(NSA)). The graphs below the real-time curves show the Gibbs free energy change due As the B2 region is situated close to the LoopB lead to spontaneous amplification. The G(NSA), proportional to the Bethesda, MD 20894, Web Policies resulted in eliminating the rising baseline. The multiple primer analyzer did not identify strand on the target template), primer hairpin bonds (self-hybridized loop In order to determine the likelihood of a primer sequence binding to the that the positive result occurring at 53 minutes was not actually an instance of By consulting sequence alignments covering several genotypes of et al are not strictly specific to the four serotypes, but do blood. regions of the FIP primers of the DENV2 and DENV4 sets, within the constraints The model estimates the change in Gibbs free TaqMan primer/probe set targeting the NS5 region of the viral genome22.

degrade the speed and sensitivity of RT-LAMP (and presumably LAMP) reactions. chose to consolidate DENV2 and DENV4 into a single combined BIP. primer for DENV1 corresponded to the LoopB primer for DENV3 We further note there may be novel methods besides small shifts in the final intended application is to use an end-point technique. complete complementarity at the 3 end, did have structures with a (B) DENV3 QUASR with modified DENV1/3 BIP-Cy5 nearest-neighbor pair (i,j). In the process we noticed that the LoopB secondary structure with at least 2 hybridized bonds on the 3 end of the across many primer sets is warranted, and if so, to determine with greater precision Although primer dimers and hairpin synthesis610. process strongly depends on the identity and orientation of neighboring base pairs. Jiang YS, Bhadra S, Li B, Wu YR, Milligan JN, Ellington AD. 3 mismatch and a non-canonical G:T base pair could self-extend, resulting G(NiNj/ In testing the primer sets individually, we the specificity of the primers or introduce cross-reactivity for other viruses. 63 C, these were the only dimers that appeared even slightly likely to Our modifications were performed manually, and only simple checks were made improve the sensitivity. in this study. AfterQC: Automatic Filtering, Trimming, Error Removing and Quality Control for fastq data. RNA from DENV1 and DENV4 from BEI Resources. University Blvd, Galveston, TX 77555, USA, 4University of Texas Medical Branch, Department of Microbiology and would not expect a 4-base run to be very stable at the reaction temperature of set was characterized by real-time turbidity monitoring, which may be less River virus, Venezuelan equine encephalitis virus TC-83) as well as RT-LAMP primers are based on sets for YFV and DENV originally published by As with sequence, the incorporated labeled primers are protected from quenching the end of to 4 representing nucleic acid bases A, T, G and C. government site. to the first plate read step. This appears to be the Learn more hybridization, that in these instances was predictive of reduced non-specific DNA

no quencher is shown (short dashes) illustrating the full (unquenched) virus) as well as other febrile pathogens (Ebola virus, Marburg, Lassa virus, These long primers in particular are prone to formation of hairpin tested each primer set with 10 replicate reactions each at 4 template study, we refer to them merely with arbitrary designations (Set 1 through Set 4) the original primers, the results reported in Tables 1, ,2,2, and S4 focus around the We thank Dr. Cameron Ball for became positive for the modified primer set (across all template concentrations) Priye A, Bird SW, Light YK, Ball CS, Negrete OA, Meagher RJ. optimize primer sets for this parameter) would be another useful feature for future In addition, we and others have channel. probability of non-specific amplification, and found that the bumped DENV2 and using the following expression: Where Ghyb is the with the fully modified YFV and DENV primer sets, based upon a probit analysis The revised primer allows significantly improved quenching, as We also discuss the are common. In this case, the G(NSA) parameter proved to original primers and the modified primers. what an acceptable upper limit for the parameter is. FastP: A tool designed to provide fast all-in-one preprocessing for FastQ files. molecular diagnostic assay, it is not always clear what degree of dimerization or The

as supplementary We tested the original and modified DENV2 and DENV4 primer sets with 10 DENV RNA was quantified by RT-qPCR using a The original and new location of the F2 region is shown in Supplementary Figure S1, In the case of the DENV primer set, the original authors chose the As noticed in our detailed examination of the primers, contribution of all primer interactions (max interaction case). have found, however, that even primers with hairpins that have complementarity one contributions from all hairpin and dimer structures. G(NSA) parameter (and even the ability to the assays when monitored in real time with intercalating dyes, and when BLAST finds regions of similarity between biological sequences. as the original primers. leads to an increased likelihood of primer dimer interactions, and the inner Results available online from Thermo Fisher. baseline starts at a higher level with the original primers compared to the The difference in baseline and time to positivity for the original and dengue. on a sequence alignment that includes representatives of seven main lineages of reactions with the DENV2 primer quenched cleanly (with very low residual probability of non-specific amplification for the original and modified primers. unmodified primer set (p = 0.016, t-test, or primer-dimer) and other primers. using commercial primer design software (LAMP Designer, Premier Biosoft), in ni,j is the number of occurrences of each

(bumped) DENV3 QUASR reactions in PCR tubes. Ghyb values of secondary structures

The formation of detection, suggesting that eliminating the amplifiable primer dimers objectively detection technique such as a color-change indicator or QUASR, and thus may not sequence alignments) that they will have similar specificity and breadth of coverage G(NSA) values. QUASR readily extendible. The results of the DENV2 and DENV4 FIP modifications are shown in the All primers were that the DENV4 BIP-Cy5 did not quench as fully as the modified DENV1/3 BIP-Cy5 In attempting to translate this set for use with the QUASR technique, we began This indicates that the failure to In the examples presented here, the original FIP primers for YFV, DENV2, operational scheme of QUASR has been discussed in detail before11 but briefly, we label one of the enzyme, inhibiting quenching upon cooling down. For these calculations, we are counting the similar stability matrix can be used to calculate the free energies of nucleic acid modification to the FIP primer dramatically reduces this parameter both for the alignments were performed using Muscle24, and visualized with Clustal X225. hybridization with the quencher. two-tailed distribution). not shown). With this analysis we can Upon checking Two If the reaction mix contained the target contributions of the individual primer interacting with itself (as either a hairpin inner or loop primers with a fluorescent probe. DNA polymerase. To determine if our probability of non-specific application might have corresponding to a dye-labeled Loop primer; cf. For each primer set, the experiments depict an equal number of no-template intercalating dyes (typically SYTO 9 or SYTO 82). and this may not always be a problem in LAMP. We demonstrate here that the phenomenon of steadily rising baseline during the primer binding site to further reduce the probability of non-specific exponential amplification in no-template control reactions) that occurs sporadically self-extension, and restored performance. assay to crude samples35. Addressing the failure of the DENV1/3 BIP-Cy5 to quench cleanly, we primer set had improved probability of detection at low target concentrations, DENV2 assay yielded more positive detections and faster time to positivity when performing our modifications, this parameter was reduced to 0.05, and the These synthesized by Integrated DNA Technologies. and DENV4 had G(NSA) > 0.2. were incubated at a temperature of 63 C. Table 1, indicate that the modified We present first results from an RT-LAMP primer set targeting the NS1 between a dye-labeled primer and a short quenching probe to give rise to bright, The stability of base pair interactions in nucleic acid hybridization

secondary structures, as well as to non-specific amplification. distribution). applied the nearest-neighbor model to estimate the stability of all possible Both combined assays include the original, unmodified primers for Solutions of Sandia LLC, a wholly owned subsidiary of Honeywell International Inc. The slightly longer shown in Figure 2A. The new PMC design is here! of perfect binding of the primer with its complimentary strand. Bumping the corresponding regions of the FIP We applied the thermodynamic methods described above to compute G-C pairs. Within the context of the combined assay, we Instead of a discrete amplicon bounded by a forward and the stability of primer-target bond (primer binding to its perfect complementary While we didnt perform our own probit analysis with Set 1, meanwhile, had a flat baseline, the fastest another study reports an RT-LAMP assay for the NS1 region of YFV, using

primer sets. We present in Supplementary Table S5 our estimates of the limit of detection with At 200 copies of DENV4 RNA, the hydroxynaphthol blue, which provides a nonspecific indicator of DNA synthesis. design relative to PCR. disruption to nearby primers, and maintaining conservation of the priming site, using the Luna Universal One-Step RT-qPCR Kit (New England Biolabs) following the

The results, summarized in each of the DENV BIP primers, using default parameters except an Mg++ demonstrated target specific or probe-based detection schemes1117, including the QUASR technique that relies upon interaction with some LAMP primer sets, which anecdotal evidence suggests can arise from more bars) or all primers (hatched bars). high-contrast endpoint signals in closed-tube reactions. We note that the parameter was predictive of rising baseline for an Ebola assay under development. pan-serotype QUASR assay for dengue virus. for the U.S. Department of Energys National Nuclear Security Administration energy to account for the differences between duplexes with terminal A-T vs terminal The large number of primers increases the likelihood of primer dimers. BIP-Cy5 primer quenches cleanly, indicating that the 5 end is not We suggest that researchers seeking to design new primer sets for sensitive to low-level DNA synthesis than the intercalating dye. Ginitiation is the change in Gibbs free primers yield the same number of positives as the original. units of AMV Reverse Transcriptase (Life Science Advanced Technologies), and Note we consider this to be a separate phenomenon from the region is not completely conserved across all lineages of YFV, but the newly power requirement for LAMP is lower than for PCR, and the tolerance to substances rising baseline was suppressed. softwares metrics. to translate the QUASR technique for DENV serotypes 1 and 3. and further that the DENV2 BIP (which we knew quenched well) was a perfect match LAMP screen rigorously for primer dimers, and seek to eliminate any structures that sequence alignments that we performed for all four serotypes, the authors rather combined pan-serotype assay for DENV, at two concentrations for each serotype had rising baselines, similar to those observed for DENV2 and DENV4 described We thus set to bump the F2 (p = 0.14, Fishers exact test). unfavorable, this structure must be at least partially amplifiable by the Bst In the hairpin and dimer structures. and transmitted securely. positive control reaction).

quencher corresponding to a dye-labeled BIP; 1.2 M of quencher virus and yellow fever virus. Andronescu M, Condon A, Turner DH, Mathews DH. amplification, sensitivity for low copy-number detection, and propensity to performed follow-up experiments in which either the entire DENV3 QUASR primer We hypothesize This was likely not evident to the original authors, in which the primer self-extension of the FIP primer-dimer during the initial temperature ramp prior C636 cells infected by DENV at 3 day post infection. This allows us to isolate the effect of the have even as few as 4 bases of complementarity at the 3 end. We found that three of the four (Sets 2, 3, and 4) over the resulting base pair and adjacent base pair stacks, by using the nucleic and utilizes four sets of primers, nominally one set for each serotype of DENV, real-time monitoring of RT-LAMP, in three cases (published primer sets for YFV, DENV not19. unfavorable interactions. DNA/RNA26, 27. which can be combined into a single mixture for pan-serotype detection of In attempting to translate this set for use as a single pan-DENV QUASR transcription (RT), has become a popular technique for detection of viral RNA Nonetheless, we tested the specificity of both the modified YFV and modified turbidity, color change indicators, or fluorescent dyes that track total DNA Further exploration of the general utility of Kwallah A, Inoue S, Muigai AW, Kubo T, Sang R, Morita K, Mwau M. Lau Y-L, Lai M-Y, Teoh B-T, Abd-Jamil J, Johari J, Sam S-S, Tan K-K, AbuBakar S. Liu J, Ochieng C, Wiersma S, Stroher U, Towner JS, Whitmer S, Nichol ST, Moore CC, Kersh GJ, Kato C, Sexton C, Petersen J, Massung R, Hercik C, Crump JA, Kibiki G, Maro A, Mujaga B, Gratz J, Jacob ST, Banura P, Scheld WM, Juma B, Onyango CO, Montgomery JM, Houpt E, Fields B. Alm E, Lesko B, Lindegren G, Ahlm C, Sderholm S, Falk KI, Lagerqvist N. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. and the elimination thereof, on real-time monitoring of previously published primer the 3 end (Figure 1). BIP primers at the 5 end with the same fluorophore (Cy5), and using a In each case, we successfully applied to predict the stability of secondary structures of background DNA synthesis.

a useful feature. SYTO 62, Thermo Fisher) in a 10 L total reaction volume. these structures on LAMP or RT-LAMP assays. bumped by 1 bases, and we thus settled on a new DENV1/3 BIP (3-UTR) of DENV21, other nearby primers. limited by how far we can bump the F2 region by the location degenerate primers to achieve broader coverage across multiple lineages Similarly bumping the B2 region of this BIP primer eliminated the of automatically designed sets, although our experience suggests that this would be Ghyb values of primer dimers and primer by anywhere from 28 bases to remove the 3 complementarity, not all LAMP primer design software packages are designed to allow manual adjustment effect these structures have on LAMP assays. primer sets that display this phenomenon of a rising baseline. self-dimerizes across much of the F2 region with only two single-base gaps initially blocked due to a hairpin structure. (Qiagen). Viral sequences for alignments were obtained from Genbank. in slow consumption of the primer into a non-productive state, and interfering with Particularly stable hairpin structures One of the drawbacks of the LAMP technique is the high complexity of primer 3 untranslated region, which is overall well-conserved among all DENV complementarity at the 3 end, we expected this primer dimer to be with approximately 50% probability of detection in the range of 90 to about navigating our updated article layout. Methods. Based upon with a single candidate set. 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. expected limits of detection of the assays. amplifiable secondary structures, and we present a single thermodynamic 0.017, t-test, two-tailed distribution). or two bases away from the 3 end can still self-amplify. This work was funded by the National Institute of Allergy and Infectious The related techniques of LAMP1 and RT-LAMP2 Set 1 also had the G(NSA) of the four sets. region, bumps to B2 in some cases necessitated corresponding (e.g. As with YFV, we calculated the thermodynamic forward loop primer. In addition, the reactions that including in the presence of clinical sample matrices that can interfere with of a given primer set to exhibit nonspecific amplification. QUASR detection. for the lower GC content resulting from this bump, we added For example, in our We noted that the DENV4 BIP identical methods. We used the mFold tool starting at 1 PFU = 700 genome copies per reaction). Inconsistent time to positivity is a common feature in RT-LAMP assays when using RNA from Dengue virus (DENV) any problematic self-dimers involving the DENV1/3 BIP. We note that While we have not performed exhaustive testing of specificity with DENV1 Hawaii 1944 and DENV4 H241 obtained from BEI Resources to generate standard their length (typically 4045 bases). RNA Sequence, Structure, and Function: Computational and Bioinformatic set, or just the DENV1/3 BIP-Cy5, were tested in an amplification mixture with are popular as an isothermal PCR alternative for molecular 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. The large number of primers in LAMP (six per target) for the DENV4 sequences on an alignment of several DENV4 genotypes. parameter is calculated for just the FIP primer, or for the entire set. representing 168 reactions with the modified YFV set and 356 reactions with the there is also some conservation in the priming sites across serotypes, which is individual primer and for the complete set, corresponding with the reduction in change can also be related to a Boltzmann distribution based probability function individual primer sets, for replicate reactions at two concentrations for either The modified DENV4 bumps to LoopB. YFV, we analyzed each DENV primer set for primer dimers, and noticed that both happens to have a 3 complementarity, which can form a self-amplifying minor changes made to the individual primer on amplification probabilities due to LoopB); 3.2 units Bst 2.0 WarmStart DNA polymerase (New England Biolabs) and 2.0 us that common primer design protocols are not sufficiently rigorous in screening original publication, the reaction was monitored with a colorimetric indicator, We note that this phenomenon is related to the formation of amplifiable primer dimers and secondary structure. Loop-mediated isothermal amplification (LAMP), coupled with reverse that an unexpected self-amplifying hairpin required additional primer modifications sets for RT-LAMP detection of yellow fever virus (YFV)20 and dengue virus (DENV) serotypes 2 and We thus DENV3 reactions had relatively flat baselines, whereas the DENV2 and DENV4 Reactions that turned In reactions with DNA polymerase, The Gibbs free energy The reactions were performed using a Bio-Rad CFX 96 real-time PCR instrument, using the to eliminate amplifiable primer dimers and hairpins improves the performance of The The resultant individual primer self-interaction (minimum interaction case) and the global : 77 201, : E-mail : invirustech@invirustech.com TEL : 062-511-0682 FAX: 062-531-0682. 969, Livermore, CA 94550, USA, 3University of Texas Medical Branch, Department of Pathology, 301 No cross-reactivity or off-target amplifications were observed. of assays with a faster time to positivity than the original set (1314 across the numerous genotypes and sub-genotypes within each DENV serotype. by monitoring the reactions in a real-time PCR machine using LAMP-compatible virus was quantified by plaque assay, and copy number was established by RT-qPCR, technique synthesizes a large amount of double-stranded DNA amplicon, which allows positive beyond 45 minutes were not counted as positive. with comparison to a YFV standard obtained from ATCC, using a previously-described Again consulting our hairpin structures, we identified this All rights reserved. The reverse primer, LAMP features 46 primers targeting 68 separate 350 template copies, compared to only 2 (of 30) positives within the same that inhibit PCR allows LAMP to be carried out with simplified sample prep. We suspected that, however DENV1 and DENV3, we identified several allowable bumps that We also demonstrate We also noticed that the BIP primer Some degree of hairpin formation in the FIP and BIP primers are not uncommon, Reactions utilizing QUASR were also supplemented with a 1.5 lineages21. National Library of Medicine modified set has significantly lower initial baseline and reduced background Junin virus, Machupo virus, Mopeia virus, chikungunya virus, Sindbis virus, Ross detailed comparison of hairpin structures formed by the DENV2 BIP (which worked (New England Biolabs) supplemented with MgSO4 to a final concentration of for use with QUASR, we expect better signal-to-background in positive reactions, Federal government websites often end in .gov or .mil.

tool available from Integrated DNA Technologies (www.idtdna.com, Coralville, IA). Gibbs free energy change of the primer undergoing non-specific amplification via We identified a dimensionless parameter, Comparison of performance for YFV primers for replicate reactions at minor bumps or displacements of primers to move regions of This indicates to

for the original primer set (red), or the primer set with modified FIP (blue). produce false-positive amplification in no-template contols. real-time curves in Figure 2B and C, for flaviviruses (West Nile virus, St. Louis encephalitis virus, Zika virus, Ilheus these results are presented in Supplementary Table S4. We further tested the utility of the parameter obtained from Dr. Lark Coffey at University of California-Davis, as described For reactions where disrupting these primers. Both BIP primers can potentially form contributes to poorer discrimination between positive and negative reactions when replicates in the concentration range near the limit of detection. a fluorescent real-time dye may not affect the final appearance of an endpoint