key: cord-313571-umcbulcw
authors: Martínez-Murcia, Antonio; Bru, Gema; Navarro, Aaron; Ros-Tárraga, Patricia; García-Sirera, Adrián; Pérez, Laura
title: 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
date: 2020-05-05
journal: bioRxiv
DOI: 10.1101/2020.04.27.065383
sha: 
doc_id: 313571
cord_uid: umcbulcw

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 (10-106 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.

. In order to illustrate the extent of mismatching of GPS™ 198 kit, an alignment of primers/probe sequences to selected SARS-CoV sequences is shown in 199 Table 2 . Standard calibration curves of the qPCR were performed from ten-fold 202 dilution series (Figure 3a Only three months ago, an outbreak of severe pneumonia caused by the novel coronavirus SARS-225

CoV-2 started in Wuhan (China) and rapidly expanded to almost all areas worldwide. Due to the 226 need of urgent detection tools, several laboratories developed RT-qPCR methods by designing 227 primers and probes from the alignment of a single-first provided SARS-CoV-2 genome sequence 228 to known SARS-CoV, and some of these protocols were published at the WHO website [12] [13] [14] [15] [16] [17] [18] [19] . 229

As the number of genomes available rapidly expanded during last January, the GPS™ CoVID-19 230 dtec-RT-qPCR Test was based on a more specific target for SARS-CoV-2 detection, being this 231 company one of the pioneers marketing a PCR-kit for the CoVID-19 worldwide. mismatches which may affect to its binding, particularly considering its short primary structure. In 246 some cases, single nucleotide mismatching was observed in some primers, but none of them were 247 located close to primer 3'-end. Considering all updated alignments, only the Australia/VIC01/2020 248 sequence showed a unique mismatch to the GPS™ probe. Therefore, a full calibration was run 249 using synthetic RNA-genomes from Australia/VIC01/2020 isolate and the resulting Ct values The in silico analysis for exclusivity was more complex, showing a wide range of discriminative 254 power for the methods subjected to analysis (Table 1) . For instance, the two RT-qPCR designs 255 IP2 and IP4 developed by Institut Pasteur seems to discriminate well between SARS-CoV-2 and 256 other respiratory virus as confirmed for a panel of specimens [14] . The CDC from Atlanta (USA) the N3 primer/probe, but a few weeks ago, this set was removed from the panel 259 CoV-2 and SARS-CoV, P2 probe was considered specific for SARS-CoV-2. Although our in silico 267 results confirmed that purpose for P1 (Table 1) , the RdRp_P2 assay may also react with some 268 other coronaviruses. The CDC in China developed two RT-qPCR assays for ORF1ab and N genes 269 (Table 2 ). The analysis standard curve was repeated a 296 minimum of ten times and average value for all parameters were optimum according to standard 297 limits. For reliability, the coefficient of variation (CV) obtained in all cases for both, repeatability 298 and reproducibility, was always much lower than 10%. The LOD was tested with the usual protocol 299 for 10 copies repeated 15 times with a positive result in all cases (100%). LOQ assays were 300 performed in two sets of 15 tests for both 10 copies of standard templates. The LOQ measurement 301 in both cases was validated with a t-Student test with a confidence interval of 95%. The kit received 302 diagnostic validation at two different reference laboratories (ISCIII, Madrid; and PHE, London). 303

The results shown in Table 3 indicated 100% of diagnostic sensitivity and 100% of diagnostic 304 specificity was assigned. Currently, the kit is being used in several Spanish hospitals and 305 diagnostic laboratories. representative calibration curve with stats. Inclusivity of the GPS™ CoVID-19 dtec-RT-qPCR Test 486 using six ranges of decimal dilution from 5·10 6 copies to 5·10 copies, and negative control. 

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