key: cord-0781281-ibpd9mrl authors: Toms, Derek; Li, Julang; Cai, Hugh Y title: Evaluation of WHO listed COVID-19 qPCR primers and probe in silico with 375 SERS-CoV-2 full genome sequences date: 2020-04-28 journal: nan DOI: 10.1101/2020.04.22.20075697 sha: 1cf8303b7c30df31ae713dd76f45c0f17be40a2f doc_id: 781281 cord_uid: ibpd9mrl Quantitative reverse-transcription PCR (qRT-PCR) assays remains the gold standard for detection of the SARS-CoV-2 virus because of its sensitivity and specificity. However, successful design of qRT-PCR assays requires accurate viral genome sequences. With mutations accumulating as the virus is transmitted globally, we sought to compare current assays recommended by the World Health Organization with available SARS-CoV-2 genomic sequences in silico. While most sequences were conserved, there were notable mismatches, particularly in assays developed using early sequences when compared to more recent isolates. We recommend that any assay being evaluated for diagnostic tests be compared with prevalent sequence data from the region of proposed testing and that continued publicly accessible sequence information continue to be provided by the research community. During infection outbreak in crisis like the COVID-19 pandemic, diagnostics are a crucial step to manage 2 the rate of infection, especially when clinical symptoms are difficult to distinguish from other respiratory 3 infections such as influenza. Public health measures decisions, such as a patient and contact tracing 4 requiring further quarantine and surveillance are intimately related to whether a suspected case has 5 been confirmed. Therefore, speed and accuracy of such tests are paramount, thus the development and 6 application of sensitive and reliable diagnostic tests are critical. 7 Among several platforms available, quantitative (real-time) reverse-transcription polymerase chain 8 reaction (qRT-PCR) remains the primary means for diagnosing the novel coronavirus SARS-CoV-2, the 9 pathogen responsible for COVID-19 (reviewed in 1). Using short DNA oligonucleotides (primers and 10 probes) that are complementary to specific sequences of viral genetic material, qRT-PCR diagnostic tests 11 thus are based on detection of the genetic material of the virus and require accurate design to ensure 12 detection sensitivity and specificity 2, 3 . Primers, one on each strand of DNA serve as starting points for 13 the DNA polymerase enzyme that carries out the RT-PCR reaction, while probes bind between the 14 primer sites and confer specificity. Design of primers and probes is based on sequenced viral genomes 15 that have been publicly available since late December 2019 and typically target regions of the open 16 reading frame (ORF) 1ab, envelope (E) and nucleocapsid (N) coding regions 4 . 17 One of our major challenges in the diagnosis of COVID-19 is that detection sensitivity and specificity of 18 SARS-CoV-2 genetic material using qRT-PCR are variable and sometimes low 5 . Multiple factors may have 19 contributed to the low sensitivity of SARS-CoV-2 detection: location of clinical sampling; low patient viral 20 load; sporadic shedding; and variation in detection kits from different manufacturers. One of the key 21 factors determining kit detection sensitivity is how efficiently primers and probes bind target genetic 22 material. This in turn is dependent on kit manufacturers using the most appropriate viral genome 23 We performed nine in silico evaluations of SARS-CoV-2 qRT-PCR primers and probes listed in the 30 protocols published on the WHO website 6 . Summarized results are shown in Table 1 The primers and probe of the confirmation E gene qPCR had 100% identity with 376/377 SARS-CoV-2 E 55 gene sequences available in GenBank. The probe had 1 bp mismatch with sequence MT039890 (isolate 56 SNU01 from a Korean patient imported from Wuhan). 57 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 28, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 28, 2020. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 28, 2020. . All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 28, 2020. . https://doi.org/10.1101/2020.04.22.20075697 doi: medRxiv preprint Potential Rapid Diagnostics, Vaccine and Therapeutics for 2019 Novel Coronavirus Considerations for accurate gene expression measurement by reverse transcription quantitative PCR when analysing clinical samples Reliability of real-time reverse-transcription PCR in clinical diagnostics: gold standard or substandard? 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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity