LitCovid-sentences  

PMC:6988269 / 8893-20325 JSONTXT 13 Projects

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Id Subject Object Predicate Lexical cue
T72 0-7 Sentence denotes Results
T73 8-150 Sentence denotes Before public release of virus sequences from cases of 2019-nCoV, we relied on social media reports announcing detection of a SARS-like virus.
T74 151-219 Sentence denotes We thus assumed that a SARS-related CoV is involved in the outbreak.
T75 220-341 Sentence denotes We downloaded all complete and partial (if > 400 nt) SARS-related virus sequences available in GenBank by 1 January 2020.
T76 342-541 Sentence denotes The list (n = 729 entries) was manually checked and artificial sequences (laboratory-derived, synthetic, etc), as well as sequence duplicates were removed, resulting in a final list of 375 sequences.
T77 542-641 Sentence denotes These sequences were aligned and the alignment was used for assay design (Supplementary Figure S1).
T78 642-800 Sentence denotes Upon release of the first 2019-nCoV sequence at virological.org, three assays were selected based on how well they matched to the 2019-nCoV genome (Figure 1).
T79 801-981 Sentence denotes The alignment was complemented by additional sequences released independently on GISAID (https://www.gisaid.org), confirming the good matching of selected primers to all sequences.
T80 982-1118 Sentence denotes Alignments of primer binding domains with 2019-nCoV, SARS-CoV as well as selected bat-associated SARS-related CoV are shown in Figure 2.
T81 1119-1229 Sentence denotes Figure 1 Relative positions of amplicon targets on the SARS coronavirus and the 2019 novel coronavirus genome
T82 1230-1400 Sentence denotes E: envelope protein gene; M: membrane protein gene; N: nucleocapsid protein gene; ORF: open reading frame; RdRp: RNA-dependent RNA polymerase gene; S: spike protein gene.
T83 1401-1487 Sentence denotes Numbers below amplicons are genome positions according to SARS-CoV, GenBank NC_004718.
T84 1488-1587 Sentence denotes Figure 2 Partial alignments of oligonucleotide binding regions, SARS-related coronaviruses (n = 9)
T85 1588-1789 Sentence denotes The panels show six available sequences of 2019-nCoV, aligned to the corresponding partial sequences of SARS-CoV strain Frankfurt 1, which can be used as a positive control for all three RT-PCR assays.
T86 1790-2059 Sentence denotes The alignment also contains a closely related bat virus (Bat SARS-related CoV isolate bat-SL-CoVZC45, GenBank accession number MG772933) as well as the most distant member within the SARS-related bat CoV clade, detected in Bulgaria (GenBank accession number NC_014470).
T87 2060-2135 Sentence denotes Dots represent identical nucleotides compared with the WH_Human_1 sequence.
T88 2136-2175 Sentence denotes Nucleotide substitutions are specified.
T89 2176-2230 Sentence denotes Blue arrows: oligonucleotides as specified in Table 1.
T90 2231-2292 Sentence denotes More comprehensive alignments can be found in the Supplement.
T91 2294-2345 Sentence denotes Assay sensitivity based on SARS coronavirus virions
T92 2346-2517 Sentence denotes To obtain a preliminary assessment of analytical sensitivity, we used purified cell culture supernatant containing SARS-CoV strain Frankfurt-1 virions grown on Vero cells.
T93 2518-2630 Sentence denotes The supernatant was ultrafiltered and thereby concentrated from a ca 20-fold volume of cell culture supernatant.
T94 2631-2770 Sentence denotes The concentration step simultaneously reduces the relative concentration of background nucleic acids such as not virion-packaged viral RNA.
T95 2771-2930 Sentence denotes The virion preparation was quantified by real-time RT-PCR using a specific in vitro-transcribed RNA quantification standard as described in Drosten et al. [8].
T96 2931-3089 Sentence denotes All assays were subjected to replicate testing in order to determine stochastic detection frequencies at each assay’s sensitivity end point (Figure 3A and B).
T97 3090-3264 Sentence denotes All assays were highly sensitive, with best results obtained for the E gene and RdRp gene assays (5.2 and 3.8 copies per reaction at 95% detection probability, respectively).
T98 3265-3317 Sentence denotes These two assays were chosen for further evaluation.
T99 3318-3522 Sentence denotes One of the laboratories participating in the external evaluation used other basic RT-PCR reagents (TaqMan Fast Virus 1-Step Master Mix) and repeated the sensitivity study, with equivalent results (E gene:
T100 3523-3555 Sentence denotes 3.2 RNA copies/reaction (95% CI:
T101 3556-3571 Sentence denotes 2.2–6.8); RdRP:
T102 3572-3604 Sentence denotes 3.7 RNA copies/reaction (95% CI:
T103 3605-3614 Sentence denotes 2.8–8.0).
T104 3615-3779 Sentence denotes Of note, the N gene assay also performed well but was not subjected to intensive further validation because it was slightly less sensitive (Supplementary Figure S2)
T105 3780-3925 Sentence denotes Figure 3 Determination of limits of detection based on SARS coronavirus genomic RNA and 2019 novel coronavirus-specific in vitro transcribed RNA
T106 3926-4008 Sentence denotes CI: confidence intervals; c/r: copies per reaction; IVT: in vitro-transcribed RNA.
T107 4009-4011 Sentence denotes A:
T108 4012-4062 Sentence denotes E gene assay, evaluated with SARS-CoV genomic RNA.
T109 4063-4065 Sentence denotes B:
T110 4066-4118 Sentence denotes RdRp gene assay evaluated with SARS-CoV genomic RNA.
T111 4119-4204 Sentence denotes C: E-gene assay, evaluated with 2019-nCoV-specific in vitro-transcribed RNA standard.
T112 4205-4207 Sentence denotes D:
T113 4208-4292 Sentence denotes RdRp gene assay evaluated with 2019-nCoV-specific in vitro-transcribed RNA standard.
T114 4293-4340 Sentence denotes The x-axis shows input RNA copies per reaction.
T115 4341-4573 Sentence denotes The y-axis shows positive results in all parallel reactions performed, squares are experimental data points resulting from replicate testing of given concentrations (x-axis) in parallels assays (eight replicate reactions per point).
T116 4574-4637 Sentence denotes Technical limits of detection are given in the panels headings.
T117 4638-4692 Sentence denotes The inner line is a probit curve (dose-response rule).
T118 4693-4727 Sentence denotes The outer dotted lines are 95% CI.
T119 4729-4827 Sentence denotes Sensitivity based on in vitro-transcribed RNA identical to 2019 novel coronavirus target sequences
T120 4828-5113 Sentence denotes Although both assays detected 2019-nCoV without polymorphisms at oligonucleotide binding sites (Figure 2), we additionally generated in vitro-transcribed RNA standards that exactly matched the sequence of 2019-nCoV for absolute quantification and studying the limit of detection (LOD).
T121 5114-5240 Sentence denotes Replicate reactions were done at concentrations around the detection end point determined in preliminary dilution experiments.
T122 5241-5394 Sentence denotes The resulting LOD from replicate tests was 3.9 copies per reaction for the E gene assay and 3.6 copies per reaction for the RdRp assay (Figure 3C and D).
T123 5395-5550 Sentence denotes These figures were close to the 95% hit rate of 2.9 copies per reaction, according to the Poisson distribution, expected when one RNA molecule is detected.
T124 5552-5628 Sentence denotes Discrimination of 2019 novel coronavirus from SARS coronavirus by RdRp assay
T125 5629-5965 Sentence denotes Following the rationale that SARS-CoV RNA can be used as a positive control for the entire laboratory procedure, thus obviating the need to handle 2019-nCoV RNA, we formulated the RdRp assay so that it contains two probes: a broad-range probe reacting with SARS-CoV and 2019-nCoV and an additional probe that reacts only with 2019-nCoV.
T126 5966-6120 Sentence denotes By limiting dilution experiments, we confirmed that both probes, whether used individually or in combination, provided the same LOD for each target virus.
T127 6121-6222 Sentence denotes The specific probe RdRP_SARSr-P2 detected only the 2019-nCoV RNA transcript but not the SARS-CoV RNA.
T128 6224-6280 Sentence denotes Detection range for SARS-related coronaviruses from bats
T129 6281-6475 Sentence denotes At present, the potential exposure to a common environmental source in early reported cases implicates the possibility of independent zoonotic infections with increased sequence variability [5].
T130 6476-6674 Sentence denotes To show that the assays can detect other bat-associated SARS-related viruses, we used the E gene assay to test six bat-derived faecal samples available from Drexler et al. [13] und Muth et al. [14].
T131 6675-6743 Sentence denotes These virus-positive samples stemmed from European rhinolophid bats.
T132 6744-6877 Sentence denotes Detection of these phylogenetic outliers within the SARS-related CoV clade suggests that all Asian viruses are likely to be detected.
T133 6878-7024 Sentence denotes This would, theoretically, ensure broad sensitivity even in case of multiple independent acquisitions of variant viruses from an animal reservoir.
T134 7026-7045 Sentence denotes Specificity testing
T135 7047-7065 Sentence denotes Chemical stability
T136 7066-7298 Sentence denotes To exclude non-specific reactivity of oligonucleotides among each other, causing artificial fluorescent signals, all assays were tested 120 times in parallel with water and no other nucleic acid except the provided oligonucleotides.
T137 7299-7359 Sentence denotes In none of these reactions was any positive signal detected.
T138 7361-7402 Sentence denotes Cross-reactivity with other coronaviruses
T139 7403-7583 Sentence denotes Cell culture supernatants containing all endemic human coronaviruses (HCoV)‑229E, ‑NL63, ‑OC43 and ‑HKU1 as well as MERS-CoV were tested in duplicate in all three assays (Table 2).
T140 7584-7665 Sentence denotes For the non-cultivable HCoV-HKU1, supernatant from human airway culture was used.
T141 7666-7840 Sentence denotes Viral RNA concentration in all samples was determined by specific real-time RT-PCRs and in vitro-transcribed RNA standards designed for absolute quantification of viral load.
T142 7841-7946 Sentence denotes Additional undiluted (but not quantified) cell culture supernatants were tested as summarised in Table 2.
T143 7947-8012 Sentence denotes These were additionally mixed into negative human sputum samples.
T144 8013-8095 Sentence denotes None of the tested viruses or virus preparations showed reactivity with any assay.
T145 8096-8282 Sentence denotes Table 2 Tests of known respiratory viruses and bacteria in clinical samples and cell culture preparations for cross-reactivity in 2019 novel coronavirus E and RdRp gene assays (n = 310)
T146 8283-8354 Sentence denotes Clinical samples with known viruses Clinical samplesa Virus isolatesb
T147 8355-8372 Sentence denotes HCoV-HKU1 14 1c
T148 8373-8390 Sentence denotes HCoV-OC43 16 2d
T149 8391-8408 Sentence denotes HCoV-NL63 14 1e
T150 8409-8426 Sentence denotes HCoV-229E 18 2f
T151 8427-8442 Sentence denotes MERS-CoV 5 1g
T152 8443-8472 Sentence denotes Influenza A(H1N1)pdm09 17 1
T153 8473-8497 Sentence denotes Influenza A(H3N2) 16 1
T154 8498-8528 Sentence denotes Influenza A (untyped) 11 NA
T155 8529-8552 Sentence denotes Influenza A(H5N1) 1 1
T156 8553-8576 Sentence denotes Influenza A(H7N9) 0 1
T157 8577-8618 Sentence denotes Influenza B (Victoria or Yamagata) 31 1
T158 8619-8649 Sentence denotes Rhinovirus/enterovirus 31 NA
T159 8650-8691 Sentence denotes Respiratory syncytial virus (A/B) 33 NA
T160 8692-8721 Sentence denotes Parainfluenza 1 virus 12 NA
T161 8722-8751 Sentence denotes Parainfluenza 2 virus 11 NA
T162 8752-8781 Sentence denotes Parainfluenza 3 virus 14 NA
T163 8782-8811 Sentence denotes Parainfluenza 4 virus 11 NA
T164 8812-8841 Sentence denotes Human metapneumovirus 16 NA
T165 8842-8859 Sentence denotes Adenovirus 13 1
T166 8860-8882 Sentence denotes Human bocavirus 6 NA
T167 8883-8898 Sentence denotes Legionella spp.
T168 8900-8905 Sentence denotes 3 NA
T169 8906-8921 Sentence denotes Mycoplasma spp.
T170 8923-8928 Sentence denotes 4 NA
T171 8929-8961 Sentence denotes Total clinical samples 297 NA
T172 8962-9095 Sentence denotes a For samples with multiple viruses detected, the virus with highest concentration is listed, as indicated by real-time PCR Ct value.
T173 9096-9161 Sentence denotes b Directly quantified or spiked in human negative-testing sputum.
T174 9162-9227 Sentence denotes c 1 × 105 RNA copies/mL, determined by specific real-time RT-PCR.
T175 9228-9274 Sentence denotes Isolated from human airway epithelial culture.
T176 9275-9356 Sentence denotes d 1 × 1010 RNA copies/mL, determined by specific real-time RT-PCR of one isolate.
T177 9357-9438 Sentence denotes The other isolate was not quantified but spiked in human negative-testing sputum.
T178 9439-9504 Sentence denotes e 4 × 109 RNA copies/mL, determined by specific real-time RT-PCR.
T179 9505-9585 Sentence denotes f 3 × 109 RNA copies/mL, determined by specific real-time RT-PCR of one isolate.
T180 9586-9663 Sentence denotes The other isolate was not quantified spiked in human negative-testing sputum.
T181 9664-9729 Sentence denotes g 1 × 108 RNA copies/mL, determined by specific real-time RT-PCR.
T182 9731-9844 Sentence denotes Exclusivity of 2019 novel coronavirus based on clinical samples pre-tested positive for other respiratory viruses
T183 9845-10106 Sentence denotes Using the E and RdRp gene assays, we tested a total of 297 clinical samples from patients with respiratory disease from the biobanks of five laboratories that provide diagnostic services (one in Germany, two in the Netherlands, one in Hong Kong, one in the UK).
T184 10107-10321 Sentence denotes We selected 198 samples from three university medical centres where patients from general and intensive care wards as well as mainly paediatric outpatient departments are seen (Germany, the Netherlands, Hong Kong).
T185 10322-10488 Sentence denotes The remaining samples were contributed by national public health services performing surveillance studies (RIVM, PHE), with samples mainly submitted by practitioners.
T186 10489-10688 Sentence denotes The samples contained the broadest range of respiratory agents possible and reflected the general spectrum of virus concentrations encountered in diagnostic laboratories in these countries (Table 2).
T187 10689-10747 Sentence denotes In total, this testing yielded no false positive outcomes.
T188 10748-10874 Sentence denotes In four individual test reactions, weak initial reactivity was seen but they were negative upon retesting with the same assay.
T189 10875-11111 Sentence denotes These signals were not associated with any particular virus, and for each virus with which initial positive reactivity occurred, there were other samples that contained the same virus at a higher concentration but did not test positive.
T190 11112-11432 Sentence denotes Given the results from the extensive technical qualification described above, it was concluded that this initial reactivity was not due to chemical instability of real-time PCR probes but most probably to handling issues caused by the rapid introduction of new diagnostic tests and controls during this evaluation study.