Validation of a novel molecular assay to the diagnostic of COVID-19 based on real time PCR with high resolution melting
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Abstract
The emergence of the COVID-19 pandemic resulted in an unprecedented need for RT-qPCR-based molecular diagnostic testing, placing a strain on the supply chain and the availability of commercially available PCR testing kits and reagents. The effect of limited molecular diagnostics-related supplies has been felt across the globe, disproportionally impacting molecular diagnostic testing in developing countries where acquisition of supplies is limited due to availability. The increasing global demand for commercial molecular diagnostic testing kits and reagents has made standard PCR assays cost prohibitive, resulting in the development of alternative approaches to detect SARS-CoV-2 in clinical specimens, circumventing the need for commercial diagnostic testing kits while mitigating the high-demand for molecular diagnostics testing. The timely availability of the complete SARS-CoV-2 genome in the beginning of the COVID-19 pandemic facilitated the rapid development and deployment of specific primers and standardized laboratory protocols for the molecular diagnosis of COVID-19. An alternative method offering a highly specific manner of detecting and genotyping pathogens within clinical specimens is based on the melting temperature differences of PCR products. This method is based on the melting temperature differences between purine and pyrimidine bases. Here, RT-qPCR assays coupled with a High Resolution Melting analysis (HRM-RTqPCR) were developed to target different regions of the SARS-CoV-2 genome (RdRp, E and N) and an internal control (human RNAse P gene). The assays were validated using synthetic sequences from the viral genome and clinical specimens (nasopharyngeal swabs, serum and saliva) of sixty-five patients with severe or moderate COVID-19 from different states within Brazil; a larger validation group than that used in the development to the commercially available TaqMan RT-qPCR assay which is considered the gold standard for COVID-19 testing. The sensitivity of the HRM-RTqPCR assays targeting the viral N, RdRp and E genes were 94.12, 98.04 and 92.16%, with 100% specificity to the 3 SARS-CoV-2 genome targets, and a diagnostic accuracy of 95.38, 98.46 and 93.85%, respectively. Thus, HRM-RTqPCR emerges as an attractive alternative and low-cost methodology for the molecular diagnosis of COVID-19 in restricted-budget laboratories.
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SciScore for 10.1101/2021.07.14.21260471: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Ethics Statement: This study was approved by the ethical committee of Clementino Fraga Filho University Hospital, from Federal University of Rio de Janeiro (
Consent: All patients or a member of their families signed the consent form.Sex as a biological variable Patients under 18 years old, pregnant women and with cancer were excluded from this study. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Real Time RT-PCR with TaqMan assays: All patient samples were analyzed for SARS-Cov-2 N2 and human RNAseP targets using a commercial One-Step RTqPCR TaqMan kit, SARS-…SciScore for 10.1101/2021.07.14.21260471: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: Ethics Statement: This study was approved by the ethical committee of Clementino Fraga Filho University Hospital, from Federal University of Rio de Janeiro (
Consent: All patients or a member of their families signed the consent form.Sex as a biological variable Patients under 18 years old, pregnant women and with cancer were excluded from this study. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Real Time RT-PCR with TaqMan assays: All patient samples were analyzed for SARS-Cov-2 N2 and human RNAseP targets using a commercial One-Step RTqPCR TaqMan kit, SARS-Cov-2 N2suggested: NoneSoftware and Algorithms Sentences Resources A primer-BLAST search (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) using the sequences of each primer pair and allowing up to 4 mismatches (none in the 3’ s end) in the complete set of the NCBI database (non-redundant sequences) resulted only in SARS-CoV-2 and RNAse P gene related-sequences. https://www.ncbi.nlm.nih.gov/tools/primer-blast/suggested: (Primer-BLAST, RRID:SCR_003095)Student’s t-test or Mann– Whitney Rank Sum test was used to analyze the statistical significance of the observed differences (according to the parametric or nonparametric distribution of the values, respectively) with SigmaPlot for Windows version 12.0 SigmaPlotsuggested: (SigmaPlot, RRID:SCR_003210)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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