In silico design and validation of commercial kit GPS™ CoVID-19 dtec-RT-qPCR Test under criteria of UNE/EN ISO 17025:2005 and ISO/IEC 15189:2012
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Abstract
Background
The Corona Virus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has become a serious infectious disease affecting human health worldwide and rapidly declared a pandemic by WHO. Early, several RT-qPCR were designed by using only the first SARS-CoV-2 genome sequence.
Objectives
A few days later, when additional SARS-CoV-2 genome were retrieved, the kit GPS™ CoVID-19 dtec-RT-qPCR Test was designed to provide a highly specific detection method and commercially available worldwide. The kit was validated following criteria recommended by the UNE/EN ISO 17025:2005 and ISO/IEC 15189:2012.
Methods
The present study approached the in silico specificity of the GPS™ CoVID-19 dtec-RT-qPCR Test and RT-qPCR designs currently published. The empirical validation parameters specificity (inclusivity/exclusivity), quantitative phase analysis (10-10 6 copies), reliability (repeatability/reproducibility) and sensitivity (detection/quantification limits) were evaluated for a minimum of 10-15 assays. Diagnostic validation was achieved by two independent reference laboratories, the Instituto de Salud Carlos III (ISCIII), (Madrid, Spain) and the Public Health England (PHE; Colindale, London, UK).
Results
The GPS™ RT-qPCR primers and probe showed the highest number of mismatches with the closet related non-SARS-CoV-2 coronavirus, including some indels. The kits passed all parameters of validation with strict acceptance criteria. Results from reference laboratories 100% correlated with these obtained by suing reference methods and received an evaluation with 100% of diagnostic sensitivity and specificity.
Conclusions
The GPS™ CoVID-19 dtec-RT-qPCR Test, available with full analytical and diagnostic validation, represents a case of efficient transfer of technology being successfully used since the pandemic was declared. The analysis suggested the GPS™ CoVID-19 dtec-RT-qPCR Test is the more exclusive by far.
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SciScore for 10.1101/2020.04.27.065383: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources GENOME SEQUENCES ALIGNMENT AND PHYLOGENETIC ANALYSIS: Partial alignments of ten SARS-CoV-2 genomic sequences and these from strains of Bat-CoV, Bat SARS-like-CoV, SARS-CoV, Pangolin-CoV (ca. 18,141 bp) and the corresponding phylogenetic tree (Figure 1) was obtained by Neighbour joining method [20], with bootstrap values for 1000 replicates, using the MEGA 5.2.2 software [21]. 2.2. MEGAsuggested: (Mega BLAST, RRID:SCR_011920)IN SILICO COMPARATIVE ANALYSIS OF PRIMERS/PROBES … SciScore for 10.1101/2020.04.27.065383: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources GENOME SEQUENCES ALIGNMENT AND PHYLOGENETIC ANALYSIS: Partial alignments of ten SARS-CoV-2 genomic sequences and these from strains of Bat-CoV, Bat SARS-like-CoV, SARS-CoV, Pangolin-CoV (ca. 18,141 bp) and the corresponding phylogenetic tree (Figure 1) was obtained by Neighbour joining method [20], with bootstrap values for 1000 replicates, using the MEGA 5.2.2 software [21]. 2.2. MEGAsuggested: (Mega BLAST, RRID:SCR_011920)IN SILICO COMPARATIVE ANALYSIS OF PRIMERS/PROBES SPECIFICITY: The primers and probes of GPS™ COVID-19 dtec-RT-qPCR Test and the RT-qPCR designs recently published [12–19] were aligned to the corresponding homologous regions of 63 SARS-CoV-2 strains and closely related Betacoronavirus using the Basic Local Alignment Search Tool (BLAST) software available on the National Center for Biotechnology Information (NCBI, https://blast.ncbi.nlm.nih.gov/Blast.cgi) website databases (Bethesda, MD, USA). BLASTsuggested: (BLASTX, RRID:SCR_001653)https://blast.ncbi.nlm.nih.gov/Blast.cgisuggested: (TBLASTX, RRID:SCR_011823)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.
- No funding statement was detected.
- No protocol registration statement was detected.
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