Comparative in silico design and validation of GPS™ CoVID-19 dtec-RT-qPCR test

Abstract

Aims: Providing a ready-to-use reverse transcriptase qPCR (RT-qPCR) method fully validated to detect the SARS-CoV-2 with a higher exclusivity than this shown by early published RT-qPCR designs. Methods and Results: The specificity of the GPS TM CoVID-19 dtec-RT-qPCR test by analysis of sequence alignments was approached and compared with other RT-qPCR designs. The GPS TM CoVID-19 dtec-RT-qPCR test was validated following criteria of UNE/EN ISO 17025:2005 and ISO/IEC 15189:2012. Diagnostic validation was achieved by two independent reference laboratories, the Instituto de Salud Carlos III, (Madrid, Spain), the Public Health England (Colindale, London, UK), and received the label CE-IVD. The GPS design showed the highest exclusivity and passed all parameters of validation with strict acceptance criteria. Results from reference laboratories 100% correlated with these obtained by using reference methods and showed 100% of diagnostic sensitivity and specificity. Conclusions: The CE-IVD GPS TM CoVID-19 dtec-RT-qPCR test, available worldwide with full analytical and diagnostic validation, is the more exclusive for SARS-CoV-2 by far. Significance and Impact of the Study: Considering the CoVID-19 pandemic status, the exclusivity of RT-qPCR tests is crucial to avoid false positives due to related coronaviruses. This work provides of a highly specific and validated RT-qPCR method for detection of SARS-CoV-2, which represents a case of efficient transfer of technology successfully used since the pandemic was declared. Background Last 30th January, the Emergency Committee of the World Health Organization (WHO) under the International Health Regulations (IHR) declared an outbreak of pneumonia, lately named Corona Virus Disease 2019 (COVID-19), as a 'Public Health Emergency of International Concern' (PHEIC). The disease is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and the first genome was rapidly provided (http://viro logical.org/t/novel-2019-coronavirus-genome/319). SARS-CoV-2 is a Betacoronavirus subgenus Sarbecovirus of group 2B, with similar characteristics than SARS-CoV,

Current quantitative polymerase chain reaction to detect severe acute respiratory syndrome coronavirus 2 may give positive results for other described coronavirus

“…: MT121216.1) for IP2, E and RdRp-P2 qPCR designs; Pangolin CoV isolate PCoV_GX-P2V, complete genome (Genbank No. : MT072864.1) for N2; and hCoV-19/pangolin/Guangxi/P1E/2017 (Accesion ID: EPI_ISL_410539) for GPS™ qPCR kit.Information about the target sequence on Pangolin CoV sequence selected of each qPCR design and the amplicons size are summarized inTable 1.These results agree with those recently published byLiu et al (2020) andMartínez-Murcia et al (2020), where Bat CoV RaTG13 and Pangolin CoV sequences showed the highest similarities with SARS-CoV-2.…”

“…RaTG13 is a single Bat-CoV sequence which shows the highest homology (96.7%) with all the SARS-CoV-2 sequences, and it was deposited the 27 th January, much after the outbreak started. Therefore, the possibility of RNA contamination during genome sequencing should be ruled-out before taking further conclusions (Martínez-Murcia et al, 2020). preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted February 9, 2021. ; https://doi.org/10.1101/2021.02.08.21251332 doi: medRxiv preprint MT121216.1; d) N2 with Pangolin CoV -MT072864.1; e) RdRp-P2 with Pangolin CoV -MT121216.1; f) E with pangolin CoV -MT121216.1; g) GPS™ CoVID-19 dtec-RT-qPCR Test with Pangolin CoV -EPI_ISL_410539.…”

“…The primers and probes sequences of the qPCR designs subjected to analysis in the present study (CDC Atlanta, 2020;Corman et al, 2020;Institut Pasteur, 2020;Martínez-Murcia et al, 2020) All rights reserved. No reuse allowed without permission.…”

“…The primers and probes sequences of the qPCR designs subjected to analysis in the present study (CDC Atlanta, 2020; Corman et al, 2020; Institut Pasteur, 2020; MartÍnez-Murcia et al, 2020) were used to find similar regions between close biological sequences 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). The closest coronavirus strain for each one, different from SARS-CoV-2, was selected and used to test their in vitro exclusivity.…”

“…However, when testing SARS-CoV-2 by qPCR in environmental samples, the exclusivity of primers/probe sequences becomes extremely relevant because the diversity of organisms in nature is huge when compared with human clinical samples, where only some selected SARS-CoV-2 clones are expected. In a previous paper, we analysed the in silico exclusivity of some of the most worldwide used qPCR designs published by WHO and our own qPCR design transferred as GPS™ CoVID-19 dtec-RT-qPCR Test and commercially available (MartÍnez-Murcia et al, 2020). The analysis showed a relatively low SARS-CoV-2 in silico specificity in several of the qPCR designs, while the qPCR available by GPS™ was the most exclusive by far.…”