{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,21]],"date-time":"2026-08-21T10:26:05Z","timestamp":1787307965587,"version":"build-2736575974"},"update-to":[{"DOI":"10.12688\/f1000research.109673.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,5,2]],"date-time":"2026-05-02T00:00:00Z","timestamp":1777680000000}},{"DOI":"10.12688\/f1000research.109673.2","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,5,2]],"date-time":"2026-05-02T00:00:00Z","timestamp":1777680000000}}],"reference-count":42,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2026,5,2]],"date-time":"2026-05-02T00:00:00Z","timestamp":1777680000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Vice-Rectory of Research and Extension of Industrial University of Santander","award":["projectNo.76900"],"award-info":[{"award-number":["projectNo.76900"]}]},{"name":"Ministry of Science, Technology, and Innovation of Colombia","award":["contractNo.369-2020"],"award-info":[{"award-number":["contractNo.369-2020"]}]},{"name":"Ministry of Science, Technology, and Innovation of Colombia","award":["code1102101576900"],"award-info":[{"award-number":["code1102101576900"]}]}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"<ns5:p>\n                    Background The COVID-19 pandemic, caused by SARS-CoV-2, highlighted the need for accurate and scalable diagnostic tools such as RT-qPCR. However, false-negative results may occur due to viral mutations and RNA secondary structures within target regions. Methods High-performance computing (HPC) was used to compile SARS-CoV-2 genomic sequences from GenBank and GISAID and generate consensus sequences for primer and probe design. A region within the\n                    <ns5:italic>ORF8<\/ns5:italic>\n                    gene was selected and evaluated alongside targets from the\n                    <ns5:italic>E<\/ns5:italic>\n                    and\n                    <ns5:italic>N<\/ns5:italic>\n                    genes and the\n                    <ns5:italic>RNase P<\/ns5:italic>\n                    control. Nasopharyngeal swab samples were collected from patients with a prior clinical diagnosis consistent with SARS-CoV-2 infection, as well as from volunteers, and total RNA was extracted using the MagMAX kit. RT-qPCR assays were performed in both single and multiplex formats. Denaturing solutions composed of tetraethylammonium chloride and dimethyl sulfoxide, as well as adjusted dNTP proportions based on viral nucleotide composition, were evaluated. Exploratory Ct-based performance metrics were estimated using predefined threshold criteria with the Caret package in R. Results A total of 126,576 SARS-CoV-2 genomes collected between January and December 2020 were used to construct a reference dataset. A target region within the\n                    <ns5:italic>ORF8<\/ns5:italic>\n                    gene exhibiting predicted secondary structures was selected for primer and probe design. Forty-nine clinical samples were analyzed, of which 22 tested positive across the evaluated gene targets. Variability in detection patterns was observed across sampling periods. The evaluated formulations were associated with changes in Ct values in both single and multiplex RT-qPCR assays, depending on the conditions and sample set. Conclusions The incorporation of denaturing solutions and the adjustment of nucleotide proportions were associated with changes in RT-qPCR performance under the evaluated experimental conditions. These findings suggest that RNA secondary structure and nucleotide composition may influence assay behavior; however, further studies are required to assess the broader applicability of this approach.\n                  <\/ns5:p>","DOI":"10.12688\/f1000research.109673.3","type":"journal-article","created":{"date-parts":[[2026,5,2]],"date-time":"2026-05-02T10:14:07Z","timestamp":1777716847000},"page":"331","update-policy":"https:\/\/doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Denaturing and dNTPs reagents improve SARS-CoV-2 detection via single and multiplex RT-qPCR"],"prefix":"10.12688","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1502-4967","authenticated-orcid":false,"given":"Cristian E.","family":"Cadena-Caballero","sequence":"first","affiliation":[{"name":"Grupo de Investigaci\u00f3n Computo Avanzado y a Gran Escala - CAGE Bucaramanga Santander 680006 Universidad Industrial de Santander"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3174-8153","authenticated-orcid":false,"given":"Lina M.","family":"Vera-Cala","sequence":"additional","affiliation":[{"name":"Grupo de Investigaci\u00f3n en Demograf\u00eda, Salud P\u00fablica y Sistemas de Salud - GUINDESS Bucaramanga Santander 680006 Universidad Industrial de Santander"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3227-8651","authenticated-orcid":false,"given":"Carlos","family":"Barrios-Hernandez","sequence":"additional","affiliation":[{"name":"Grupo de Investigaci\u00f3n Computo Avanzado y a Gran Escala - CAGE Bucaramanga Santander 680006 Universidad Industrial de Santander"},{"name":"Centro de Supercomputaci\u00f3n y C\u00e1lculo Cient\u00edfico de la Universidad Industrial de Santander -SC3UIS Bucaramanga Santander 680006 Universidad Industrial de Santander"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2818-3323","authenticated-orcid":false,"given":"Diego","family":"Rueda-Plata","sequence":"additional","affiliation":[{"name":"Grupo de Investigaci\u00f3n Computo Avanzado y a Gran Escala - CAGE Bucaramanga Santander 680006 Universidad Industrial de Santander"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1645-9986","authenticated-orcid":false,"given":"Lizeth J.","family":"Forero-Buitrago","sequence":"additional","affiliation":[{"name":"Grupo de Investigaci\u00f3n Computo Avanzado y a Gran Escala - CAGE Bucaramanga Santander 680006 Universidad Industrial de Santander"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3088-5255","authenticated-orcid":false,"given":"Carolina S.","family":"Torres-Jimenez","sequence":"additional","affiliation":[{"name":"Grupo de Investigaci\u00f3n Computo Avanzado y a Gran Escala - CAGE Bucaramanga Santander 680006 Universidad Industrial de Santander"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erika","family":"Lizarazo-Gutierrez","sequence":"additional","affiliation":[{"name":"Grupo de Investigaci\u00f3n Computo Avanzado y a Gran Escala - CAGE Bucaramanga Santander 680006 Universidad Industrial de Santander"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9908-099X","authenticated-orcid":false,"given":"Mayra","family":"Agudelo-Rodriguez","sequence":"additional","affiliation":[{"name":"Grupo de Investigaci\u00f3n Computo Avanzado y a Gran Escala - CAGE Bucaramanga Santander 680006 Universidad Industrial de Santander"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1668-7671","authenticated-orcid":false,"given":"Francisco","family":"Martinez-Perez","sequence":"additional","affiliation":[{"name":"Grupo de Investigaci\u00f3n Computo Avanzado y a Gran Escala - CAGE Bucaramanga Santander 680006 Universidad Industrial de Santander"},{"name":"Centro de Supercomputaci\u00f3n y C\u00e1lculo Cient\u00edfico de la Universidad Industrial de Santander -SC3UIS Bucaramanga Santander 680006 Universidad Industrial de Santander"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2026,5,2]]},"reference":[{"key":"ref41","doi-asserted-by":"publisher","first-page":"S1341-321X","DOI":"10.1016\/j.jiac.2024.02.032","article-title":"Clinical evaluation of a modified SARS-CoV-2 rapid molecular assay, ID NOW TM COVID-19 2.0.","volume":"24","author":"Y Arakawa","year":"2024","journal-title":"J. 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Beggs, Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, 16 May 2022, version 1, 1 approved with reservations","URL":"https:\/\/f1000research.com\/articles\/11-331\/v1#referee-response-135398","order":0,"name":"referee-response-135398","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Francisco Martinez-Perez<\/b>; \n<i>Posted: 14 Mar 2024<\/i>; \n<b>Reviewer 1.<\/b>  \n<b>Andrew D. Beggs<\/b> \n<b>Institute of Cancer and Genomic Sciences, University of Birmingham, Birmingham, UK.<\/b>  The authors have set out to design a new primer set to target the \n<i>ORF8<\/i> gene region of the SARS-CoV-2 genome, as well as understand the effect of denaturing agents on the efficiency of detection by reducing secondary structures and increasing RT-qPCR. It is certainly an interesting approach. However, I have a few comments and suggestions to further improve the manuscript.  \n<b>Response: We greatly appreciate the comments, suggestions and corrections made by reviewer 1, which greatly enriched the work.<\/b>  1.&nbsp;The abstract is confusing. They state that they design \n<i>ORF8<\/i> primers using \n<i>E <\/i>and \n<i>N<\/i> as targets but what they actually mean is that they use these as references. Also, they state that the denaturing agents reduce secondary structure and increase efficiency but don't really get the results of this across in the abstract.  \n<b>Response: The correction is accepted. The abstract of the manuscript is modified and expanded, providing clarity in the methods and results sections.<\/b>  \n<b>Abstract<\/b> \n<b>Background: <\/b>The COVID-19 pandemic, caused by the SARS-CoV-2, can be effectively managed with diagnostic tools such as RT-qPCR. However, it can produce false-negative results due to viral mutations and RNA secondary structures from the target gene sequence. \n<b>Methods: <\/b>With High Performance Computing, the complete SARS-CoV-2 genome was obtained from the GenBank\/GISAID to generate consensus sequences to design primers\/probes for RT-qPCR. \n<i>ORF8<\/i> gene was selected, meanwhile, \n<i>E<\/i> and \n<i>N<\/i> and \n<i>RNAse P<\/i> were according to CDC protocol. Nasopharyngeal swab samples were collected from patients diagnosed with SARS-CoV-2. Total RNA was purified according MagMAX kit, it was used in single, and multiplex RT-qPCR. To avoid templated secondary structures, compensate nucleotide proportions and improve Ct values, a solution composed of tetraethylammonium chloride and dimethyl sulfoxide and other with corresponding to dNTPs proportions in accordance SARS-CoV-2 genome were included. Sensitivity and specificity according to Ct values were determined with the Caret package in R software. \n<b>Results: <\/b>126,576 SARS-CoV-2 genomes from January to December 2020 comprised a database. From this, a region near of 5' \n<i>ORF8<\/i> gene showed three stem-loops was used for primers\/FAM-probe. 49 samples were obtained, from them, 22 were positive to gene selected regions. Interestingly, samples from October to November 2020 were positive for all regions, however, in January 2021 different results were observed in \n<i>ORF8<\/i>. An improvement in Ct with the adjuvant solutions was determined in all samples with others SARS-CoV-2 primers\/probes, for both single and multiplex RT-qPCR. The inclusion of the denaturing solution in single RT-qPCR increased its sensitivity with respect to the commercial method, while in multiplex the opposite was generated. \n<b>Conclusions: <\/b>Including adjuvant solutions to prevent the formation of RNA secondary structures and the adjustment of the nucleotide ratios of SARS-CoV-2 improved single and multiplex RT-qPCR for viral identification, demonstrating its potential application in health public.  2. They talk in detail about the characterization in the GISAID\/GenBank databases, but don't explain well that the reason they are doing this is to help design the ORF8 gene probes. This should be clarified.  \n<b>Response: Clarity is provided in the methods to use of databases for <i>ORF8<\/i> design.<\/b>  Due to the appearance of mutations in several genes of the SARS-CoV-2 reference genome that could generate false negatives with the primers and probes authorized for identification by RT-qPCR. A region coding for the accessory protein ORF8 was selected, hypothesizing that it did not have a high mutation frequency. Therefore, monthly consensus sequences were generated to determine its identity pattern with respect to new genomes.  3.&nbsp;The primer\/probe concentrations are not given but should be to increase the reproducibility of the experiments.  \n<b>Response: The concentration in nmole of the primers\/probes is reported below. It could also be included in Table 1.<\/b>  4.&nbsp;A 2xn table of the primer sets with a set threshold in positive\/negative and quoted sensitivity, specificity etc. would greatly aid interpretation of the data.  \n<b>Response: The calculation of the sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of the viral identification tests of the participants was included in the manuscript.<\/b>  \n<b>Methodology <\/b> \n<b>Calculation of sensitivity, specificity and predictive values of SARS-CoV-2<\/b>\n<b>.<\/b> To calculate the sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) of each of the reactions with the single and multiplex primers used, a script was implemented in R using the Caret package to calculate the values generated by each RT-qPCR with their respective primers\/probes (https:\/\/github.com\/GenomicUIS\/Sensitivity-specificity-PPV-and-NPV-for-SARS-CoV-2.git) (Kuhn, 2008; R Core Team 2020). Cycle threshold (Ct) was defined for each of the primers in the single reactions of each of the processed samples, where, the value of 18-35 Ct was considered true positive, Ct 35 &gt; false positive, Ct 18 &lt; true negative. Subsequently, the Cts values obtained were pooled and evaluated per sample. Therefore, if the result generated by a set of primers\/probes was at the above-mentioned threshold, it was considered a true positive identification. Whereas, results outside the threshold were considered false negative and indeterminate. Cts were identical for multiplex RT-qPCR reactions, but the results were evaluated as a whole.  R Core Team. R: A language and environment for statistical computing. \n<i>R Foundation for Statistical Computing<\/i>. 2020; Vienna, Austria. URL https:\/\/www.R-project.org\/.  Kuhn M: Building Predictive Models in R Using the caret Package. \n<i>J Stat Softw<\/i>. 2008; 28(5): 1\u201326. Doi: 10.18637\/jss.v028.i05  \n<b>Results<\/b> \n<b>Sensitivity, specificity and predictive values of SARS-CoV-2<\/b> The performance of single and multiplex RT-qPCRs with the solutions used showed that single denaturing was more sensitive than the commercial solution, but the latter is 33% more specific than the former. As for the multiplex reactions, the commercial solution showed non-significant difference in sensitivity with respect to the denaturing solution. Similarly, the specificity of the single reactions was better for the commercial solution with respect to the denaturing solution, but in the multiplex reactions this difference was not evident in the detection of true negatives as both solutions yielded results of 0. dNTPs were not evaluated due to the number of positive results (Table 4).  \n<b>Discussion<\/b>  The reagents and methods described herein allow for an immediate, facile, and cost-effective detection of the SARS-CoV-2 virus; which was demonstrated by comparing the Cts of the positive results used to determine sensitivity and specificity with those published for other countries (Arakawa \n<i>et al.,<\/i> 2024; Aranha \n<i>et al<\/i>., 2021; Chen \n<i>et al<\/i>., 2022).  Arakawa Y, Nishida Y, Sakanashi D, \n<i>et al<\/i>.: Clinical evaluation of a modified SARS-CoV-2 rapid molecular assay, ID NOW \n<sup>TM<\/sup> COVID-19 2.0. \n<i>J. Infect. Chemother<\/i>. 2024; S1341-321X(24): 00073-4. Doi: 10.1016\/j.jiac.2024.02.032.  Aranha C, Patel V, Bhor V, \n<i>et al<\/i>.: Cycle threshold values in RT-PCR to determine dynamics of SARS-CoV-2 viral load: An approach to reduce the isolation period for COVID-19 patients. \n<i>J. Med. Virol<\/i>. 2021; 93(12): 6794-6797. Doi: 10.1002\/jmv.27206.  Chen YY, Shen X, Wang YJ, \n<i>et al<\/i>.: Evaluation of the cycle threshold values of RT-PCR for SARS-CoV-2 in COVID-19 patients in predicting epidemic dynamics and monitoring surface contamination. \n<i>J. Infect. Public. Health<\/i>. 2022; 15(12): 1494-1496. Doi: 10.10 16\/j.jiph.2022.11.012.  Based on our findings, we concluded that multiplex RT-qPCR is an optimal solution for the aforementioned limitations, even though the Cts used to establish sensitivity and specificity did not show a difference in some samples between commercial methods compared to the one proposed here.  5. The authors state in the discussion that &quot;our study is the first to demonstrate the use of three genes both independently or in multiplex reactions to detect the SARS-CoV-2 virus&quot; -the Thermo Taqpath assay uses ORF, N, and S genes for detection and so is multiplex.  \n<b>Response: To our knowledge, at the time the experiments for this study were conducted (19 September 2020 to 30 January 2021), there was no kit that performed viral identification with three genes.<\/b>  According to the official website of Thermo Fisher Scientific, the Taqpath kit is based on sequences obtained from GenBank and GISAID as of 7 May 2021 (https:\/\/www.thermofisher.com\/co\/en\/home\/clinical\/clinical-genomics\/pathogen-detection-solutions\/covid-19-sars-cov-2\/multiplex.html), and subsequently obtained a letter of approval for use by the Food and Drug Administration (FDA) on 18 May 2022 (https:\/\/www.fda.gov\/media\/136113\/download). Our public database was generated by HPC between January and December 2020 (https:\/\/zenodo.org\/records\/6337537).  6.&nbsp;In the discussion of their analysis of global SARS-CoV-2 mutations they discuss &quot;thousands of millions&quot; of mutations. This makes it seem like there have been this number of mutations rather than reports of mutations, as there will be genomes submitted that have overlapping mutations between genomes. Some clarification on language would be useful.  \n<b>Response: The correction is accepted, and clarity is given in the manuscript.<\/b>  Our analysis of the global SARS-CoV-2 mutation patterns based on the GenBank and GISAID databases indicated that most mutations began in March 2020, and subsequently increased significantly, relative to the SARS-CoV-2 reference genome (GenBank access: NC_045512.2).  7.&nbsp;In addition, a discussion of why they chose to target the \n<i>ORF8<\/i> gene would be useful. I assume it is because of the relatively low mutational rate here, but it would be nice to see this explained.  \n<b>Response: Correction accepted.<\/b>  Using the SARS-CoV-2 (NC_045512) reference genome and consensus sequences from January to April 2020 (Zhu et al., 2020), an approximately 150-base pair (bp) region of the ORF8 gene was selected based on the secondary structure of its RNA transcript, which in turn was predicted using the algorithms proposed by Zuker (Zuker &amp; Jacobson, 1998) via the Mflod software (RRID:SCR_001360) (Zuker, 2003). Thus, the last set containing the codons of the central region of the \n<i>ORF8<\/i> gene, which encodes the secretion protein ORF8, was chosen because it allows proper viral adhesion to the host cell (Chan et al., 2020).  Chan JF, Kok KH, Zhu Z, \n<i>et al<\/i>.: Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan. \n<i>Emerg. Microbes. Infect<\/i>. 2020; 9(1): 221-236. DOI: 10.1080\/22221751.2020.1719902.","URL":"https:\/\/f1000research.com\/articles\/11-331\/v3#referee-comment-11276","order":1,"name":"referee-comment-11276","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.198314.r481060, Juan Sebastian Quintero Barbosa, University of Virginia, Charlottesville, Virginia, 05 May 2026, version 3, indexed","URL":"https:\/\/f1000research.com\/articles\/11-331\/v3#referee-response-481060","order":2,"name":"referee-response-481060","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.198314.r500887, Lina Silva-Trujillo, Oswaldo Cruz Institute, Rio de Janeiro, 17 Jul 2026, version 3, indexed","URL":"https:\/\/f1000research.com\/articles\/11-331\/v3#referee-response-500887","order":3,"name":"referee-response-500887","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Francisco Martinez-Perez<\/b>; \n<i>Posted: 01 Aug 2026<\/i>; \n<b>Dear Reviewer 3,<\/b>  We appreciate your valuable comments and wish to inform you that all points have been fully addressed.  Regarding the number of genomes analyzed, the total count and the source databases are mentioned in the first paragraph of the Results section. To assist the reader, we have included a link to the GitHub repository, where the process is described in detail. Additionally, at the end of that paragraph, we indicate the calculation used to obtain the total number of genomes.  Furthermore, we modified the paragraph titled &quot;\n<i>Preparation of denaturing solutions and adjustment of dNTP concentrations<\/i>&quot; to provide greater clarity regarding the volumes used in the solutions, while maintaining the concentrations described in the &quot;\n<i>SARS-CoV-2 single and multiplex RT-qPCR<\/i>&quot; section. Likewise, in the Discussion section, we explain the rationale for choosing the nucleotide composition of the viral genome, and to reinforce this concept, we have included the citation for Bong et al. (2024), which has been added to the manuscript's reference section.  Additionally, as the reviewer pointed out, to help readers interpret these findings, we have added the \u0394Ct values corresponding to the denaturing and dNTP conditions to Table 3, normalized against the commercial control.  In this new version, we have also updated the repository containing the R scripts designed to determine specificity and sensitivity, including detailed usage information to facilitate the reproducibility of the methodology for readers. The link is:&nbsp;https:\/\/github.com\/GenomicUIS\/Sensitivity-specificity-PPV-and-NPV-for-SARS-CoV-2.git.  Finally, Table 1 has been modified to include the purification specifications for the primers\/probes used in the RT-qPCR, as provided by the manufacturer, as well as the complete chemical composition of the probes (including the fluorophore, quencher, and additional modifications). Information not reported by the manufacturer is identified as &quot;NR&quot;.  We remain available for any further observations and thank you again for your time and dedication in reviewing our work.","URL":"https:\/\/f1000research.com\/articles\/11-331\/v3#referee-comment-16824","order":4,"name":"referee-comment-16824","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This research was supported by the Ministry of Science, Technology, and Innovation of Colombia (MinCiencias) [contract No. 369-2020, code 1102101576900] and by the Vice-Rectory of Research and Extension of Industrial University of Santander [project No. 76900]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":5,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}