LitCovid-sentences  

PMC:7045880 / 3166-7857 JSONTXT 13 Projects

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Id Subject Object Predicate Lexical cue
T31 0-21 Sentence denotes Materials and Methods
T32 23-25 Sentence denotes 1.
T33 26-63 Sentence denotes Clinical specimens and RNA extraction
T34 64-227 Sentence denotes Nasopharyngeal and oropharyngeal swab and sputum samples were collected from symptomatic patients to detect SARS-CoV-2 by real-time reverse transcriptase (RT)-PCR.
T35 228-369 Sentence denotes RNA was extracted from clinical samples with a QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions.
T36 370-533 Sentence denotes All specimens were handled under a biosafety cabinet according to laboratory biosafety guidelines of Korea Centers for Disease Control and Prevention for COVID-19.
T37 535-537 Sentence denotes 2.
T38 538-554 Sentence denotes Real-time RT-PCR
T39 555-712 Sentence denotes The optimal concentration of primers and probes, which were synthesized using a published sequence [12], was determined with the RNA transcripts of SARS-CoV.
T40 713-802 Sentence denotes The primer and probe sequences used for RNA-dependent RNA polymerase gene detection were:
T41 803-1019 Sentence denotes 5′-GTGARATGGTCATGTGTGGCGG-3′ (Forward), 5′-CARATGTTAAASACACTATTAGCATA-3′ (Reverse) and 5′-CAGGTGGAACCTCATCAGGAGATGC-3′ (Probe in 5-FAM/3′-BHQ format) and the primer and probe sequences used for E gene detection were:
T42 1020-1171 Sentence denotes 5′-ACAGGTACGTTAATAGTTAATAGCGT-3′ (Forward), 5′-ATATTGCAGCAGTACGCACACA-3′ (Reverse) and 5′-ACACTAGCCATCCTTACTGCGCTTCG-3′ (Probe in 5-FAM/3′-BHQ format).
T43 1172-1460 Sentence denotes A 25-μL reaction was setup that contained 5 μL of RNA, 12.5 μL of 2 × reaction buffer provided with the Agpath IDTM 1 step RT-PCR system (Thermo Fisher Scientific, Waltham, USA), 1 μL of 25 × enzyme mixture, 1 μL of forward and reverse primers at 10 pM, and 0.5 μL of each probe at 10 pM.
T44 1461-1598 Sentence denotes Reverse transcription was performed at 50°C for 30 minutes, followed by inactivation of the reverse transcriptase at 95°C for 10 minutes.
T45 1599-1752 Sentence denotes PCR amplification was performed with 40 cycles at 95°C for 15 seconds and 60°C for 1 minute using an ABI 7500 Fast instrument (Thermo Fisher Scientific).
T46 1754-1756 Sentence denotes 3.
T47 1757-1772 Sentence denotes Virus isolation
T48 1773-1874 Sentence denotes The virus was isolated from nasopharyngeal and oropharyngeal samples from putative COVID-19 patients.
T49 1875-2120 Sentence denotes Oropharyngeal samples were diluted with viral transfer medium containing nasopharyngeal swabs and antibiotics (Nystadin, penicillin-streptomycin 1:1 dilution) at 1:4 ratio and incubated for 1 hour at 4°C, before being inoculated onto Vero cells.
T50 2121-2289 Sentence denotes Inoculated Vero cells were cultured at 37°C, 5% CO2 in 1× Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2% fetal bovine serum and penicillin-streptomycin.
T51 2290-2405 Sentence denotes Virus replication and isolation were confirmed through cytopathic effects, gene detection, and electron microscopy.
T52 2406-2577 Sentence denotes Viral culture of SARS-CoV-2 was conducted in a biosafety Level-3 facility according to laboratory biosafety guidelines of Korea Centers for Disease Control and Prevention.
T53 2579-2581 Sentence denotes 4.
T54 2582-2636 Sentence denotes Next generation sequencing of viral full-length genome
T55 2637-2770 Sentence denotes Using reverse transcriptase, cDNA was synthesized from RNA extracted from the cultured cell medium in which the virus was replicated.
T56 2771-2976 Sentence denotes A next generation sequencing (NGS) library was constructed after amplifying the full-length genes of the isolates using the synthesized cDNA and primers designed based on published SARS-CoV-2 DNA sequence.
T57 2977-3042 Sentence denotes The prepared library was purified and analyzed with Miseq 150 PE.
T58 3043-3144 Sentence denotes De novo assembly was performed on the sequenced product using Megahit to secure a full-length genome.
T59 3146-3148 Sentence denotes 5.
T60 3149-3168 Sentence denotes Sequencing analysis
T61 3169-3230 Sentence denotes Gene sequencing was performed using CLC Main Workbench 7.9.1.
T62 3231-3388 Sentence denotes Alignment was conducted using human and animal coronavirus sequences registered in Global Initiative on Sharing All Influenza Data (GISAID) and NCBI GenBank.
T63 3389-3567 Sentence denotes The phylogenetic tree was analyzed using MEGA6 with the neighbor-joining method, maximum composite likelihood-parameter distance matrix, and bootstrap values of 1,000 replicates.
T64 3569-3571 Sentence denotes 6.
T65 3572-3604 Sentence denotes Transmission electron microscopy
T66 3605-3832 Sentence denotes For transmission electron microscopy, the inoculated cells were prefixed by incubating in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) to prevent the autolysis of the cells infected with virus.
T67 3833-4020 Sentence denotes To minimize the chemical reaction between pre- and post-fixation, the slides were washed 3 times using the same buffer as in the fixative solution and post-fixed with 1% osmium tetroxide.
T68 4021-4122 Sentence denotes After washing 3 times with deionized water, en bloc staining was performed using 0.5% uranyl acetate.
T69 4123-4287 Sentence denotes Thereafter, 30%, 50%, 70%, 80%, 90%, and 100% ethanol were used sequentially in ascending concentration for dehydration, which was substituted with propylene oxide.
T70 4288-4381 Sentence denotes The slides were then embedded in Epon812 plastic resin, and polymerized at 70°C for 48 hours.
T71 4382-4546 Sentence denotes The prepared plastic block was cut to 70-nm thick sections using an ultramicrotome and mounted on a 100-mesh nickel grid, and electrostained with 5% uranyl acetate.
T72 4547-4691 Sentence denotes The sections were observed with a transmission electron microscope (Libra120, Carl Zeiss, Germany) at an acceleration voltage of 120 kV [13–15].