LitCovid-sentences  

PubMed:32535302 JSONTXT 14 Projects

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Id Subject Object Predicate Lexical cue
T1 0-153 Sentence denotes Multiple assays in a real-time RT-PCR SARS-CoV-2 panel can mitigate the risk of loss of sensitivity by new genomic variants during the COVID-19 outbreak.
T2 154-165 Sentence denotes OBJECTIVES:
T3 166-347 Sentence denotes In this study, five SARS-CoV-2 PCR assay panels were evaluated against the accumulated genetic variability of the virus to assess the effect on sensitivity of the individual assays.
T4 348-366 Sentence denotes DESIGN OR METHODS:
T5 367-495 Sentence denotes As of week 21, 2020, the complete set of available SARS-CoV-2 genomes from GISAID and GenBank databases were used in this study.
T6 496-718 Sentence denotes SARS-CoV-2 primer sequences from publicly available panels (WHO, CDC, NMDC, and HKU) and QIAstat-Dx were included in the alignment, and accumulated genetic variability affecting any oligonucleotide annealing was annotated.
T7 719-727 Sentence denotes RESULTS:
T8 728-826 Sentence denotes A total of 11,627 (34.38%) genomes included single mutations affecting annealing of any PCR assay.
T9 827-1028 Sentence denotes Variations in 8,773 (25.94%) genomes were considered as high risk, whereas additional 2,854 (8.43%) genomes presented low frequent single mutations and were predicted to yield no impact on sensitivity.
T10 1029-1228 Sentence denotes In case of the QIAstat-Dx SARS-CoV-2 Panel, 99.11% of the genomes matched with a 100% coverage all oligonucleotides, and critical variations were tested in vitro corroborating no loss of sensitivity.
T11 1229-1241 Sentence denotes CONCLUSIONS:
T12 1242-1704 Sentence denotes This analysis stresses the importance of targeting more than one region in the viral genome for SARS-CoV-2 detection to mitigate the risk of loss of sensitivity due to the unknown mutation rate during this SARS-CoV-2 outbreak.INTRODUCTIONSince the beginning of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak in December 2019 (Zhu et al., 2020), the number of confirmed cases has been rising dramatically (The Open Science Prize, 2020).
T13 1705-2203 Sentence denotes According to data from the World Health Organization (WHO), Coronavirus Disease 2019 or COVID-19 (Gorbalenya et al., 2020) has been diagnosed in over 6,057,853 people from more than 200 countries or territories and caused over 371,166 deaths worldwide as of 1 June 2020 (World Health Organization, 2020a).SARS-CoV-2 is the seventh coronavirus known to infect humans and it appears to have its origin from zoonotic transmission similar to SARS-CoV (2002) and MERS-CoV (2012) (Andersen et al., 2020).
T14 2204-2356 Sentence denotes SARS-CoV-2 is hypothesised to be the product of recombination or mutation from a genetically related SARS-CoV species hosted by bats (Sun et al., 2020).
T15 2357-2575 Sentence denotes Unlike seasonal human coronaviruses HKU1, NL63, OC43 and 229E which are associated with mild symptoms (Corman et al., 2018), SARS-CoV-2 can cause pneumonia and severe acute respiratory syndrome (Andersen et al., 2020).
T16 2576-2682 Sentence denotes All coronaviruses are single, positive-stranded RNA viruses with similar genomic ∼30kb size and structure.
T17 2683-3311 Sentence denotes These genes encode non-structural proteins (ORF1a and ORF1b) involved in replication at the 5'-end and multiple structural proteins (spike (S), envelope (E), membrane (M) and nucleocapsid (N)) downstream that make up the virus particle (Chan et al., 2020; Wu et al., 2020).During novel RNA viral outbreaks, detection of the virus relies on real-time reverse transcription polymerase chain reaction (real-time RT-PCR) for detection of the RNA of the virus In the case of SARS-CoV-2, RT-PCR is used for confirmatory diagnosis of symptomatic patients as well as increasingly screening of asymptomatic contacts and subjects at risk.
T18 3312-3793 Sentence denotes The unprecedented outbreak posed the challenge of evaluating the different diagnostic RT-PCR assays sensitivity and specificity without a previous established gold standard.Since the availability of the first SARS-CoV-2 genomic sequence (Wu et al., 2020), several studies have reported a rapid genetic evolution of SARS-CoV-2 through a phylogenetic tree tracking the geographical spread of the virus (Andersen et al., 2020; The Open Science Prize, 2020; Wu et al., 2020; Yi, 2020).
T19 3794-4063 Sentence denotes As previously established for RNA viruses, the nucleotide mutation rate of SARS-CoV-2 is estimated to be 8E-04 substitutions per site per year as a consequence of the lack of proofreading activity of polymerases (Lauring and Andino, 2020; The Open Science Prize, 2020).
T20 4064-4608 Sentence denotes It evinces that new genetic variations of SARS-CoV-2 that will most likely occur during this evolving outbreak could compromise the sensitivity and specificity of RT-PCR detection (Lippi et al., 2020).Specifically, both natural mutation rate of a virus (Lauring and Andino, 2020) and active viral recombination (Yi, 2020) could potentially impair the efficiency of oligonucleotide annealing (Lippi et al., 2020), affecting sensitivity (increasing the limit of detection) or inclusivity (missed detection of some strains due to genomic changes).
T21 4609-4882 Sentence denotes We hypothesize that in order to minimize the risk of sensitivity loss of RT-PCR for detecting novel viruses with unknown genetic variability like SARS-CoV-2, published RT-PCR design strategies for SARS-CoV-2 detection should target more than one region in the viral genome.
T22 4883-5341 Sentence denotes This was the general approach followed to obtain the earliest available SARS-CoV-2 RT-PCR assays in panels used worldwide (Centers for Disease Control and Prevention, 2020a, 2020b; Chu et al., 2020; Corman et al., 2020; National Microbiology Data Center, 2020; QIAstat-Dx Respiratory SARS-CoV-2 Panel, 2020; University of Hong Kong, 2020; World Health Organization, 2020b), including the syndromic multiplex QIAstat-Dx Respiratory SARS-CoV-2 Panel (Table 1).
T23 5342-5831 Sentence denotes The selected conserved target regions from available SARS-CoV-2 genomes minimizes the potential cross-reactivity with other coronaviruses (Gorbalenya et al., 2020; Zhu et al., 2020), including human coronaviruses (Wu et al., 2020) and bat-SARS-like coronaviruses (Chan et al., 2020).The goal of this study is to assess how the genetic variability observed in the SARS-CoV-2 genome as of week 21, 2020 has affected sensitivity of publicly available SARS-CoV-2 real-time RT-PCR Panel assays.
T24 5832-6216 Sentence denotes Additionally, a detailed characterization and evaluation of the impact on performance of the SARS-CoV-2 assays in the QIAstat-Dx Respiratory SARS-CoV-2 Panel was carried out.METHODSThe complete set of available SARS-CoV-2 genomes larger than 26,000 nucleotides from GISAID (https://www.gisaid.org/) and GenBank (http://www.ncbi.nlm.nih.gov/genbank/) databases were used in this study.
T25 6217-6492 Sentence denotes As of week 21, 2020, a total of 30,090 and 3,729 available genomes (GISAID and GenBank respectively, Table S1) were aligned using ClustalW algorithm implemented in Geneious software v.10.0.3. (http://www.geneious.com), with a gap open and extent cost of 5 and 3 respectively.
T26 6493-6778 Sentence denotes The output alignment was curated manually.SARS-CoV-2 primer sequences from publicly available panels (WHO, CDC, NMDC, and HKU) and QIAstat-Dx (Table 1) were included in the alignment, and accumulated genetic variability affecting any oligonucleotide annealing was annotated (Table S2).
T27 6779-7089 Sentence denotes Variations were considered as high risk when they were placed in the three last nucleotides of the 3'-end, affecting the PCR elongation step (Stadhouders et al., 2010; Whiley and Sloots, 2005) or in the 5'-end of the probe affecting the natural 5'- exonuclease activity of the polymerases (Smith et al., 2002).
T28 7090-7786 Sentence denotes In addition, were also considered as high risk those variations with a significant presence (>1%) among all published genomes that could become more extended worldwide in the current or potential future spread episodes.RESULTSResults showed large nucleotide insertions (EPI_ISL_416672, EPI_ISL_418061, EPI_ISL_418066, EPI_ISL_416720, EPI_ISL_423027, EPI_ISL_424274, EPI_ISL_424327, EPI_ISL_426134, EPI_ISL_427267, EPI_ISL_429646, EPI_ISL_430896, EPI_ISL_434554, EPI_ISL_437867, EPI_ISL_445217) or fragment gene inversions (EPI_ISL_427289, EPI_ISL_427291) mainly in the polyprotein Orf1ab gene, typically indicating genetic recombination common in coronavirus genomes (Sun et al., 2020; Yi, 2020).
T29 7787-8038 Sentence denotes Since none of them were placed in binding regions of any of the PCR assays, recombination analysis was considered out of the scope of this current study.A total of 11,627 (34.38%) genomes included single mutations affecting annealing of any PCR assay.
T30 8039-9342 Sentence denotes Variations in 8,773 (25.94%) genomes were considered as high risk: a) a trinucleotide variation (GGG to AAC) present in 8,251 of the 8,257 (24.41%) genomes affecting the binding of the N gene NMDC assay forward primer; b) different single variations in the last three positions of the 3'-end or 5'-end (primers and probes respectively)..Genetic variations affecting annealing of more than one oligonucleotide of the same assay were not frequent: a) a total of 126 out of the 128 genomes with a non-critical mismatch in Reverse primer of the WHO RdRp assay (Table 2) present any other variation in the Probe (RdRp_SARSr-P2) or in the Forward primer binding regions, b) 17 out of the 8,251 genomes with multiple three consecutive mismatches at the 5'-end (GGG to AAC) of the NMDC N assay presented additional single variations in the Probe or Reverse primer binding regions; c) five genome sequences (EPI_ISL_444736 for the WHO N assay; EPI_ISL_437536, EPI_ISL_422983 and MT293178 for the CDC N1 assay; and EPI_ISL_446918 for the CDC N2 assay) presented combinations of non-critical mismatches among different oligonucleotides of the same set.Additional 2,854 (8.44%) genomes presented low frequent single mutations considered to be low risk (Table 2) and were predicted to yield no impact on sensitivity.
T31 9343-9477 Sentence denotes However, the effect in every PCR performance must be evaluated individually based on the corresponding PCR thermal profile conditions.
T32 9478-9748 Sentence denotes The impact of non-3 terminal mismatches should not be underestimated, especially in a diagnostic environment (Stadhouders et al., 2010; Whiley and Sloots, 2005).A large number of genomes with variations affecting the CDC N3 assay were also found (546 variations, 1.61%).
T33 9749-10130 Sentence denotes Although they cannot be classified as critical based on the parameters defined in the present study, this data supports why the initial N3 CDC assay was removed from the official panel due to inconclusive results (Centers of Disease Control and Prevention, 2020a, 2020b).This same method of evaluation was carried out with the QIAstat-Dx SARS-CoV-2 Panel oligonucleotide sequences.
T34 10131-10445 Sentence denotes The SARS-CoV-2 assay was originally developed as a real-time RT-PCR duplex assay covering two highly conserved target genes (RdRp and E) reported with the same fluorophore and showing a verified combined limit of detection (LoD) of 500 copies/mL in clinical samples (QIAstat-Dx Respiratory SARS-CoV-2 Panel, 2020).
T35 10446-10615 Sentence denotes A total of 33,518 (99.11 %) of 33,819 matched with a 100% coverage all oligonucleotides included in the SARS-CoV-2 assays of the QIAstat-Dx Respiratory SARS-CoV-2 Panel.
T36 10616-10830 Sentence denotes In contrast, 301 (0.89%) genomes showed a single variation affecting annealing for any oligonucleotide, and 35 (0.10%) of those 301 genomes contained a variation in any critical position of any primer or probe end.
T37 10831-10984 Sentence denotes Among these latter ones, the most frequent variation consisted of a mismatch in the last position of the 3'-end of one of the primers (7 genomes, 0.02%).
T38 10985-11183 Sentence denotes Because a mismatch in this position might impair the elongation PCR step (Stadhouders et al., 2010; Whiley and Sloots, 2005), this genomic mutation was further tested in vitro re-evaluating the LoD.
T39 11184-11579 Sentence denotes A total of twenty replicates of quantified synthetic dsDNA reproducing the sequence containing this mutation were run at the LoD concentration (500 copies/mL) described in the publicly available QIAstat-Dx Respiratory SARS-CoV-2 Panel instructions for use (QIAstat-Dx Respiratory SARS-CoV-2 Panel, 2020), and 19 out of the 20 (95%) replicates were positive (Table S3) confirming the defined LoD.
T40 11580-12034 Sentence denotes Therefore, the QIAstat-Dx SARS-CoV-2 assay remains highly specific and sensitive against SARS-CoV-2 even in light of genomic variations from the first three months of the COVID-19 outbreak (Visseaux et al., 2020).DISCUSSIONGenetic variability observed among SARS-CoV-2 genomes available until week 15, 2020 after the onset of the outbreak showed single nucleotide mutations affecting the annealing of all SARS-CoV-2 RT-PCR panels evaluated in this study.
T41 12035-12202 Sentence denotes The majority of the annotated single nucleotide variations are predicted to have no effect on sensitivity at panel level, including those placed in critical positions.
T42 12203-12603 Sentence denotes This was confirmed for the QIAstat-Dx SARS-CoV-2 Panel to show no effect on the LoD of the panel.Given that genetic variability in the SARS-CoV-2 genome is expected to increase based on the natural viral mutation and recombination rates, our results show that combination of more than one assay target in real-time RT-PCR SARS-CoV-2 panels can mitigate the risk of loss of sensitivity or specificity.
T43 12604-12915 Sentence denotes In this regards, continuous monitoring of genomic variations is essential to provide a rapid response in case assay re-design is needed.AcknowledgementsWe thank all those who have contributed sequences to the GISAID database (https://www.gisaid.org/) and to NCBI database (http://www.ncbi.nlm.nih.gov/genbank/).
T44 12916-13140 Sentence denotes We also thank the QIAstat-Dx Barcelona team for their support in their corresponding roles.Conflict of interests: LP, RP, MR, SNR, MJF,DM,MLF and JP are QIAGEN employees.No ethical approval was required to conduct this work.
T45 13141-13290 Sentence denotes The work described in this paper was funded by the employer of the authors (QIAGEN).ReferencesAndersen KG, Rambaut A, Lipkin WI, Holmes EC, Garry RF.
T46 13291-13325 Sentence denotes The proximal origin of SARS-CoV-2.
T47 13326-13431 Sentence denotes Nat Med 2020. https://doi.org/10.1038/s41591-020-0820-9.Centers for Disease Control and Prevention (CDC).
T48 13432-13510 Sentence denotes 2019-novel coronavirus (2019-nCoV) real-time rRT-PCR panel primers and probes.
T49 13511-13527 Sentence denotes 2020a; Accessed:
T50 13528-13540 Sentence denotes 09 Apr 2020.
T51 13541-13689 Sentence denotes Available from: https://www.cdc.gov/coronavirus/2019-ncov/downloads/rt-pcr-panel-primer-probes.pdf.Centers for Disease Control and Prevention (CDC).
T52 13690-13763 Sentence denotes CDC 2019-novel coronavirus (2019-nCoV) real-time RT-PCR diagnostic panel.
T53 13764-13780 Sentence denotes 2020b; Accessed:
T54 13781-13793 Sentence denotes 09 Apr 2020.
T55 13794-13906 Sentence denotes Available from: https://www.fda.gov/media/134922/download.Chan JFW, Kok KH, Zhu Z, Chu H, To KKW, Yuan S, et al.
T56 13907-14048 Sentence denotes Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan.
T57 14049-14079 Sentence denotes Emerg Microbes Infect 2020; 9:
T58 14080-14195 Sentence denotes 221-36. https://doi.org/10.1080/22221751.2020.1719902.Chu DKV, Pan Y, Cheng SMS, Hui KPY, Krishnan P, Liu Y, et al.
T59 14196-14284 Sentence denotes Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia.
T60 14285-14419 Sentence denotes Clin Chem 2020; 0(0):1-7. https://doi.org/10.1093/clinchem/hvaa029.Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, et al.
T61 14420-14486 Sentence denotes Detection of 2019 novel coronavirus (2019-nCoV) by realtimeRT-PCR.
T62 14487-14623 Sentence denotes Euro Surveill 2020; 25(3): pii=2000045. https://doi.org/10.2807/1560-7917.ES.2020.25.3.2000045.Corman VM, Muth D, Niemeyer D, Drosten C.
T63 14624-14673 Sentence denotes Hosts and sources of endemic human coronaviruses.
T64 14674-14706 Sentence denotes Adv Virus Res 2018; 100:163-188.
T65 14707-14846 Sentence denotes ISSN 0065-3527. https://doi.org/10.1016/bs.aivir.2018.01.001.Gorbalenya AE, Baker SC, Baric RS, de Groot RJ, Drosten C, Gulyaeva AA, et al.
T66 14847-14961 Sentence denotes The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2.
T67 14962-14984 Sentence denotes Nat Microbiol 2020; 5:
T68 14985-15056 Sentence denotes 536-44. https://doi.org/10.1038/s41564-020-0695-z.Lauring AS, Andino R.
T69 15057-15109 Sentence denotes Quasispecies Theory and the Behavior of RNA Viruses.
T70 15110-15217 Sentence denotes PLoS Pathog 2010; 6(7): e1001005. https://doi:10.1371/journal.ppat.1001005.Lippi G, Simundic AM, Plebani M.
T71 15218-15340 Sentence denotes Potential preanalytical and analytical vulnerabilities in the laboratory diagnosis of coronavirus disease 2019 (COVID-19).
T72 15341-15488 Sentence denotes Clin Chem Lab Med 2020; 20200285, eISSN 1437-4331, ISSN 1434-6621. https://doi.org/10.1515/cclm-2020-0285.National Microbiology Data Center (NMDC).
T73 15489-15563 Sentence denotes Novel coronavirus national science and technology resource service system.
T74 15564-15579 Sentence denotes 2020; Accessed:
T75 15580-15593 Sentence denotes 09 Apr 2020..
T76 15594-15673 Sentence denotes Available from: http://nmdc.cn/nCov/en.QIAstat-Dx Respiratory SARS-CoV-2 Panel.
T77 15674-15746 Sentence denotes QIAstat-Dx Respiratory SARS-CoV-2 Panel instructions for use (Handbook).
T78 15747-15762 Sentence denotes 2020; Accessed:
T79 15763-15775 Sentence denotes 09 Apr 2020.
T80 15776-15872 Sentence denotes Available from: https://qiastat-dx.com/row/qiastat-dx-sars-cov-2/.Smith S, Vigilant L, Morin PA.
T81 15873-15970 Sentence denotes The effects of sequence length and oligonucleotide mismatches on 5' exonuclease assay efficiency.
T82 15971-15989 Sentence denotes Nucleic Acids Res.
T83 15990-16109 Sentence denotes 2002; 30(20): e111. https://doi.org/10.1093/nar/gnf110.Stadhouders R, Pas SD, Anber J, Voermans J, Mes THM, Schutten M.
T84 16110-16232 Sentence denotes The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5' nuclease assay.
T85 16233-16257 Sentence denotes J Mol Diagn 2010; 12(1):
T86 16258-16358 Sentence denotes 109-117. https://doi.org/10.2353/jmoldx.2010.090035.Sun J, He WT, Wang L, Lai A, Ji X, Zhai X et al.
T87 16359-16368 Sentence denotes COVID-19:
T88 16369-16430 Sentence denotes Epidemiology, Evolution, and Cross-Disciplinary Perspectives.
T89 16431-16442 Sentence denotes Trends Mol.
T90 16443-16528 Sentence denotes Med0 2020; 1550. https://doi.org/10.1016/j.molmed.2020.02.008.The Open Science Prize.
T91 16529-16540 Sentence denotes Nextstrain:
T92 16541-16582 Sentence denotes Real-time tracking of pathogen evolution.
T93 16583-16625 Sentence denotes Genomic epidemiology of novel coronavirus.
T94 16626-16641 Sentence denotes 2020; Accessed:
T95 16642-16654 Sentence denotes 09 Apr 2020.
T96 16655-16758 Sentence denotes Available from: https://nextstrain.org/ncov.The University of Hong Kong (HKU), School of Public Health.
T97 16759-16842 Sentence denotes Detection of 2019 novel coronavirus (2019-nCoV) in suspected human cases by RT-PCR.
T98 16843-16858 Sentence denotes 2020; Accessed:
T99 16859-16871 Sentence denotes 09 Apr 2020.
T100 16872-17066 Sentence denotes Available from: https://www.who.int/docs/default-source/coronaviruse/peiris-protocol-16-1-20.pdf?sfvrsn=af1aac73_4.Visseaux B, Le Hingrat Q, Collin G, Bouzid D, Lebourgeois S, Le Pluart D et al.
T101 17067-17198 Sentence denotes Evaluation of the QIAstat-Dx Respiratory SARS-CoV-2 Panel, the first rapid multiplex PCR commercial assay for SARS-CoV-2 detection.
T102 17199-17295 Sentence denotes J Clin Microbiol 2020;JCM.00630-20. https//doi.org/10.1128/JCM.00630-20.Whiley DM and Sloots TP.
T103 17296-17380 Sentence denotes Sequence variation in primer targets affects the accuracy of viral quantitative PCR.
T104 17381-17403 Sentence denotes J Clin Virol 2005; 34:
T105 17404-17487 Sentence denotes 104-107. https://doi.org/10.1016/j.jcv.2005.02.010.World Health Organization (WHO).
T106 17488-17545 Sentence denotes Coronavirus disease 2019 (COVID-19) situation report -79.
T107 17546-17562 Sentence denotes 2020a; Accessed:
T108 17563-17575 Sentence denotes 09 Apr 2020.
T109 17576-17707 Sentence denotes Available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/.World Health Organization (WHO).
T110 17708-17792 Sentence denotes Laboratory testing for coronavirus disease 2019 (COVID-19) in suspected human cases.
T111 17793-17809 Sentence denotes 2020b; Accessed:
T112 17810-17822 Sentence denotes 09 Apr 2020.
T113 17823-17950 Sentence denotes Available from: https://apps.who.int/iris/rest/bitstreams/1271387/retrieve.Wu F, Zhao S, Yu B, Chen YM, Wang W, Song ZG, et al.
T114 17951-18020 Sentence denotes A new coronavirus associated with human respiratory disease in China.
T115 18021-18038 Sentence denotes Nature 2020; 579:
T116 18039-18095 Sentence denotes 265-269. https://doi.org/10.1038/s41586-020-2008-3.Yi H.
T117 18096-18154 Sentence denotes 2019 novel coronavirus is undergoing active recombination.
T118 18155-18353 Sentence denotes Clin Infect Dis 2020; ciaa219. https://doi.org/10.1093/cid/ciaa219.Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R, Niu P, Zhan F, Ma X, Wang D, Xu W, Wu G, Gao GF, Tan W.
T119 18354-18418 Sentence denotes A novel coronavirus from patients with pneumonia in China, 2019.
T120 18419-18445 Sentence denotes N Engl J Med 2020; 382(8):
T121 18446-18516 Sentence denotes 727-33. https://doi.org/10.1056/NEJMoa2001017.Authors contributionsLP:
T122 18517-18638 Sentence denotes Planned experiments, conceptualised the laboratory work, conceptualised the overall study, wrote the manuscript draft.MR:
T123 18639-18676 Sentence denotes Planned and conducted experiments.RP:
T124 18677-18714 Sentence denotes Planned and conducted experiments.SR:
T125 18715-18785 Sentence denotes Contributed to overall study planning and review of the manuscript.MJ:
T126 18786-18856 Sentence denotes Contributed to overall study planning and review of the manuscript.DM:
T127 18857-18937 Sentence denotes Contributed to overall study conceptualization and review of the manuscript.MLF:
T128 18938-19008 Sentence denotes Contributed to overall study planning and review of the manuscript.JP:
T129 19009-19092 Sentence denotes Contributed to overall study conceptualization, co-wrote of the manuscript.Table 1.
T130 19093-19354 Sentence denotes Description of earliest available SARS-CoV-2 RT-PCR assaysRT-qPCR panelAssays includedTarget genesDescriptionfReferencesWHO panela3RdRp, E, Nthe E gene assay is used as the firstline screening tool, then followed by confirmatory testing with an RdRp gene assay.
T131 19355-19600 Sentence denotes The N gene assay can eventually be analyzed as an additional confirmatory assay.Corman et al., 2020; World Health Organization, 2020bCDC (US) panelb2NThis panel contains two monoplex assays (N1, N2) designed for specific detection of SARS-CoV-2.
T132 19601-19935 Sentence denotes A specimen is considered positive for 2019-nCoV if both assays are positive, whereas if only one assay resulted positive the result is inconclusive and it should be retested.Centers of Disease Control and Prevention, 2020a, 2020bNMDC panelc2RdRp, NThis panel contains two monoplex assays designed for specific detection of SARS-CoV-2.
T133 19936-20841 Sentence denotes A specimen is considered positive for 2019-nCoV if both assays are positive.National Microbiology Data Center, 2020HKU paneld2RdRp, NThe N gene RT-PCR is recommended as a screening assay and the Orf1b assay as a confirmatory one (the N gene assay is about 10 times more sensitive than the ORF-1b gene assay in detecting positive clinical specimens).Chu et al., 2020; The University of Hong Kong, 2020.QIAstat-Dx Panele2RdRp, EThis panel contains a Duplex assay in the same optical channel to report an additive performance (including CT and endpoint fluorescence values) of the two PCR assays and it discriminates SARS-CoV-2 detection from more than other 20 viruses and bacteria responsible for upper respiratory infections including other endemic coronavirusesQIAstat-Dx Respiratoyry SARS-CoV-2 Panel, 2020RdRp: RNA-dependent RNA polymerase gene (inside the Orf1ab polyprotein gene), E: envelop gene, N:
T134 20842-22100 Sentence denotes Nucleocaspide gene.This panel was originally proposed by the Charité-Universitätsmedizin Berlin Institute of Virology (Corman et al., 2020), and then endorsed by the WHO (World Helath Organization, 2020b).Initial CDC panel contained one additional assay (N3) designed for universal detection of SARS-like coronaviruses (Centers of Disease Control and Prevention, 2020a) but has been removed due to inconclusive results based on low performance (Centers of Disease Control and Prevention, 2020b).National Microbiology Data Center (NMDC) works in collaboration with National Institute for Viral Disease Control and Prevention under Chinese Center for Disease Control and Prevention.The School of Public Health of the University of Hong Kong (HKU) developed the assay (Chu et al., 2020) and it was later established as official protocol under the WHO Network Laboratories (The University of Hong Kong, 2020).QIAstat-Dx Panel is a syndromic Point-of-Care system incorporating sample purification and real time RT-qPCR amplification for more than 20 viral and bacterial respiratory pathogens, including SARS-CoV-2.WHO and HKU assays are reactive with coronaviruses under the subgenus Sarbecovirus that includes 2019-nCoV, SARS-CoV and bat SARS-like coronaviruses.
T135 22101-22295 Sentence denotes Since SARS was eliminated in humans since 2004, individuals with samples that are positive in these RT-PCR assays should be infected by the 2019-nCoV or its related animal coronaviruses.Table 2.
T136 22296-24044 Sentence denotes Summary of genomic variants detected in complementary binding regions of earliest available SARS-CoV-2 RT-PCR assays.RT-qPCR panelassayTypeOligonucleotide nameNumber of genomes (%) with non-critical single variationsNumber of genomes (%) with high risk single variationsWHO panelEFwE_Sarbeco_F10 (0.030%)2 (0.006%)aPE_Sarbeco_P134 (0.101%)9 (0.027%)bRevE_Sarbeco_R14 (0.4041%)-RdRpFwRdRp_SARSr-F95 (0.281%)5 (0.015%)cPRdRp_SARSr-P1126 (0.373%)2 (0.006%)dPRdRp_SARSr-P215 (0.044%)-RevRdRp_SARSr-R128 (0.378%)-NFwN_Sarbeco_F70 (0.207%)4 (0.012%)ePN_Sarbeco_P169 (0.204%)11 (0.033%)fRevN_Sarbeco_R119 (0.352%)1 (0.003%)gCDC (US) panelN1Fw2019-nCoV_N1-F40 (0.118%)3 (0.009%)hP2019-nCoV_N1-P53 (0.157%)353 (1.044%)iRev2019-nCoV_N1-R168 (0.497%)4 (0.012%)jN2Fw2019-nCoV_N2-F58 (0.172%)-P2019-nCoV_N2-P102 (0.302%)41 (0.121%)kRev2019-nCoV_N2-R41 (0.121%)-N3Fw2019-nCoV_N3-F344 (1.017%)l-P2019-nCoV_N3-P107 (0.316%)6 (0.018%)mRev2019-nCoV_N3-R86 (0.254%)3 (0.009%)nNMDC panelRdRpFwForward primer23 (0.068%)-PFluorescent probe48 (0.142%)10 (0.030%)oRevReverse primer30 (0.089%)1 (0.003%)pNFwForward primer337 (0.996%)8257 (24.415%)qPFluorescent probe15 (0.044%)3 (0.009%)rRevReverse primer56 (0.166%)5 (0.015%)sHKU panelRdRpFwHKU-ORF1b-nsp14F84 (0.248%)5 (0.015%)PHKU-ORF1b-nsp14P45 (0.133%)-RevHKU-ORF1b-nsp14R62 (0.183%)2 (0.006%)uNFwHKU-NF73 (0.216%)3 (0.009%)vPHKU-NP53 (0.157%)6 (0.018%)wRevHKU-NR83 (0.245%)2 (0.006%)xQIAstat-Dx PanelRdRpFwForward primer15 (0.044%)1 (0.003%)yPFluorescent probe133 (0.393%)12 (0.035%)zRevReverse primer27 (0.080%)3 (0.009%)aaEFwForward primer57 (0.169%)11 (0.032%)bbPFluorescent probe10 (0.030%)1 (0.003%)ccRevReverse primer24 (0.071%)7 (0.021%)ddTOTALS2,854 (8.434%)8,773 (25,941%)ACUMMULATED TOTAL11,627 (34.380%)Fw:
T137 24045-24063 Sentence denotes Forward primer, P:
T138 24064-24075 Sentence denotes Probe, Rev:
T139 24076-25764 Sentence denotes Reverse primerTwo genome sequences with a mismatch in in the three last positions of the 3'-end (U to C and G to respectively).Nine genomes with a mismatch in the last three positions of the 5'-end (five A to G, and four C to U).Five genomes with a mismatch in the last three positions of the 3'-end (G to A, C to U, or G to U).Two genomes with multiple mismatches in the binding position of this probe (positions 1 (C to U), 7 (G to A), 10 (A to G), 12 (R to C), 21 (U to A) of the 5'-end)).Four genomes with a mismatch in any of the three last positions at the 3'-end (C to U, or U to C).Eleven genomes with a mismatch in position 2 of the 5'-end (C to U).One genome with a mismatch in the first position of the 3'-end (G to C).Three genomes with a mismatch in the second position of the 3'-end (A to C/G).A total of 353 genomes containing a mismatch in any of the last three positions of the 5'-end (C to A/U, C to U/G).Four genomes with a mismatch in the third position of the 3'-end (C to A).A total of 41 genomes with a mismatch in the first position of the 5'-end (A to U).A total of 292 genomes present the same mismatch in position 15 of the 3'-end (U to C).Six genomes with a mismatch in second (Y to G) or third (C to U) positions of the 5'-end.Three genomes with a mismatch in first (G to A) or second (U to A) positions of the 3'-end.Ten genomes with any mismatch in the last three positions of the 5'-end (C to A, C to U, G to A).A single genome with a mismatch in the second position of the 3'-end (G to A).A total of 8251 genomes with multiple three consecutive mismatches in positions 20,21,22 of the 3'-end, corresponding to positions 1, 2, 3 of the 5'-end (GGG to AAC).
T140 25765-27040 Sentence denotes Six additional genomes with mismatches in any of the three last positions of the 3'-end.Three genomes with a mismatch in the second (U to C) or third (G to U) positions of the 5'-end.Five genomes with a mismatch in the first (G to A) or third (C to A/U) positions of the 3'-end.Five genomes with a mismatch in the second (C to U) or third (C to U) positions of the 3'-end.Two genomes with a mismatch in the second (U to C) or third (C to U) positions of the 3'-end.Three genomes with a mismatch in the third (U to C) position of the 3'-end.Six genomes with a mismatch in the second (C to A) or third (A to G) positions of the 5'-end.Two genomes with a mismatch in the second (U to C) or third (A to G) positions of the 3'-end.One mismatch in the third position of the 3'-end.Twelve genomes with a single mismatch in any of the three last positions of the 5'-end.Three genomes with a mismatch in the second position of the 3'-end.Seven genomes with a mismatch in the last position of the 3'-end, together with four additional genomes with a mismatch in the second or third positions.A single genome with a mismatch in the second position of the 5'-end.Six genomes with a mismatch in third position of the 3'-end, and one additional genome with a double mismatch in position 1.