LitCovid-sentences  

PMC:7068163 / 661-15070 JSONTXT 13 Projects

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Id Subject Object Predicate Lexical cue
T6 0-101 Sentence denotes At the end of December 2019, an outbreak caused by a novel coronavirus was announced in Wuhan, China.
T7 102-278 Sentence denotes Since then, the number of cases has increased, especially in China but also in other countries, and public health authorities are in need to rapidly implement diagnostic tools.
T8 279-465 Sentence denotes In this paper, we describe our laboratory experiences with the novel real-time RT-PCR assays comparing different one-step PCR systems and a commercial kit, using a Bio-Rad CFX 96 cycler.
T9 467-528 Sentence denotes Timely implementation of molecular diagnostics for SARS-CoV-2
T10 529-928 Sentence denotes In connection with the ongoing outbreak of a novel coronavirus in the province Hubei and surrounding areas in China, it was expected that Europe would also be confronted with the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as infections in travellers in several Asian countries outside of China were confirmed shortly after the announcement of the outbreak in Wuhan [1-4].
T11 929-1095 Sentence denotes Therefore, it was necessary to rapidly implement adequately quick and sensitive diagnostic assays for outbreak management of SARS-CoV-2 in public health laboratories.
T12 1096-1267 Sentence denotes As soon as the World Health Organization (WHO) published the first protocols for real-time RT-PCR assays, the Bavarian Food and Health Authority started to implement them.
T13 1268-1406 Sentence denotes We ordered control material and oligonucleotides (see details below) in week 4 and ran our first SARS-CoV-2 assays on 27 January (week 5).
T14 1407-1514 Sentence denotes On the same day, the first German case of coronavirus disease 2019 (COVID-19) was diagnosed in Bavaria [1].
T15 1515-1676 Sentence denotes In the following days, health authorities implemented comprehensive measures to prevent further transmission of SARS-CoV-2, including testing of contact persons.
T16 1677-1842 Sentence denotes We initially used the protocol based on the E gene and RdRp gene developed by the German Consiliary Laboratory for Coronaviruses hosted at the Charité in Berlin [5].
T17 1843-2040 Sentence denotes Real-time RT-PCR was initially performed with the QuantiTect Virus +Rox Vial kit (QIAGEN, Hilden, Germany) on the Bio-Rad CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Feldkirchen, Germany).
T18 2041-2163 Sentence denotes The kit was chosen for its frequent and successful use in our laboratory with other assays and its immediate availability.
T19 2164-2256 Sentence denotes Primers and probes were used as described [5] and provided by TIB Molbiol (Berlin, Germany).
T20 2257-2483 Sentence denotes Control material was ordered from the European Virus Archive (EVAg) and consisted of synthetic Wuhan coronavirus 2019 E gene control (reference number 026N-03866) and SARS-CoV Frankfurt 1 RNA (reference number 004N-02005) [6].
T21 2484-2622 Sentence denotes In addition, the control of LightMix Modular Wuhan CoV RdRP-gene (TibMolbiol, Berlin, Germany) was used for the SARS-CoV-2 specific assay.
T22 2623-2724 Sentence denotes Respiratory samples (nasopharyngeal swabs or sputum) were obtained from patients and contact persons.
T23 2725-2793 Sentence denotes Sputum samples were diluted in 2 mL phosphate buffered saline (PBS).
T24 2794-2913 Sentence denotes RNA was extracted using the QIAamp Bio Robot kit (QIAGEN) on a Hamilton Microlab Star (Hamilton, Bonaduz, Switzerland).
T25 2914-3232 Sentence denotes Each sample was tested by three PCRs: the screening assay for the E gene and two confirmatory assays targeting the RdRp gene, all performed as recommended in [5], with either both probes (RdRP_SARSr-P1 and RdRP_SARSr-P2), detecting SARS-CoV and SARS-CoV-2, or only with the SARS-CoV-2-specific probe RdRP_SARSr-P2 [5].
T26 3233-3289 Sentence denotes We used human RNAse P as control for RNA extraction [7].
T27 3290-3357 Sentence denotes The cumulative numbers of all tested samples are shown in Figure 1.
T28 3358-3506 Sentence denotes Two weeks after starting the SARS-CoV-2 diagnostics, we had analysed 669 respiratory samples at the Bavarian Health and Food Safety Authority (LGL).
T29 3507-3568 Sentence denotes Among them, seven showed positive results for SARS CoV-2 RNA.
T30 3569-3632 Sentence denotes By 11 February, 14 COVID-19 cases had been detected in Bavaria.
T31 3633-3742 Sentence denotes Figure 1 Cumulative numbers of suspected COVID-19 samples tested during week 5 and 6 2020, Bavaria (n = 675)
T32 3743-3832 Sentence denotes COVID: coronavirus disease; PHEIC: public health emergency of international concern; LGL:
T33 3833-3880 Sentence denotes Bavarian Health and Food Safety Authority; WHO:
T34 3881-3907 Sentence denotes World Health Organization.
T35 3908-4047 Sentence denotes The figure shows all samples tested at LGL, negative and positive, as well as six positive samples tested in other laboratories in Bavaria.
T36 4048-4116 Sentence denotes All data are cumulative, with a final total of positive tests of 13.
T37 4118-4155 Sentence denotes Comparison of one-step RT-PCR systems
T38 4156-4328 Sentence denotes During the 2 weeks after the first PCR test on 27 January, a rapidly increasing number of case contacts in Bavaria was identified and their respiratory samples were tested.
T39 4329-4664 Sentence denotes We found that the SARS-CoV E gene screening assay with the QuantiTect Virus +Rox Vial kit showed moderate to high amounts of unspecific signals in late cycles in 61% (451/743) of the tested patient samples and also of negative extraction and non-template controls (Table, Figure 2), which complicated the evaluation of the qPCR result.
T40 4665-4745 Sentence denotes The RdRp assays were basically free from such unspecific signals in late cycles.
T41 4746-4884 Sentence denotes Cycle threshold (Ct) values of the control SARS-CoV Frankfurt 1 RNA were reached three cycles earlier in E gene assay than in RdRp assays.
T42 4885-5074 Sentence denotes Table Comparison of two different one-step real-time RT-PCR systems with SARS-CoV-2 assays from Corman et al. [5] and a commercial test kit with kit-specific assays, Bavaria, February 2020
T43 5075-5284 Sentence denotes Real-time RT-PCR system PCR efficiency (%)a, linearity (R2) Limit of detection (copies/reaction) Unspecific signals count in E gene assay in totalb Unspecific signals in E gene assay (%)b Run time (hours)
T44 5285-5389 Sentence denotes QuantiTect Virus +Rox Vial kit (QIAGEN) ND ND 451/743(75/126 NC, 376/617 patient samples) 60.7 1:50
T45 5390-5541 Sentence denotes SuperScript III One-step RT-PCR System with Platinum TaqDNA Polymerase (Invitrogen) 95 / 0,99c 50c 13/257(2/38 NC,11/219 patient samples) 5.1 1:28
T46 5542-5651 Sentence denotes RealStar SARS-CoV-2 RT-PCR kit 1.0 (Altona) 125 / 0,97d 10d 0/111(0/38 NC, 0/73 patients samples) 0 2:15
T47 5652-5701 Sentence denotes NC: negative control samples; ND: not determined.
T48 5702-5723 Sentence denotes a E = 10−1/slope − 1.
T49 5724-5944 Sentence denotes b Indicated counts and percentage values of unspecific background signals in the SARS-CoV E gene assay are based on the total number of tested patient samples as well as the negative extraction and non-template controls.
T50 5945-6076 Sentence denotes c Only for RdRp gene assays, tested with four replicates of SARS-CoV Frankfurt 1 RNA [6]; 10-fold serial dilutions were determined.
T51 6077-6118 Sentence denotes For the E gene, the assay was not linear.
T52 6119-6300 Sentence denotes d Only for the E gene, tested with two replicates of synthetic Wuhan coronavirus 2019 E gene control and SARS-CoV Frankfurt 1 RNA each [6]; 10-fold serial dilutions were determined.
T53 6301-6434 Sentence denotes Figure 2 Example image of real-time RT-PCR curves of the E gene assay with unspecific signals at late cycles, Bavaria, February 2020
T54 6435-6468 Sentence denotes RFU: relative fluorescence units.
T55 6469-6476 Sentence denotes Curves:
T56 6477-6479 Sentence denotes 1:
T57 6480-6674 Sentence denotes Wuhan coronavirus 2019 E gene positive control; 2: SARS-CoV Frankfurt 1 RNA positive control; 3,4,6,8: negative patient samples; 5: extraction negative control; 7: non-template negative control.
T58 6675-6725 Sentence denotes Signal is given in log scale with threshold = 200.
T59 6726-6821 Sentence denotes PCR was performed with SuperScript III system and E gene primers and probe as published in [5].
T60 6822-6889 Sentence denotes Curves of positive controls (1 and 2) show expected sigmoid curves.
T61 6890-6955 Sentence denotes Curves 3–6 show unspecific signals with increase above threshold.
T62 6956-7032 Sentence denotes Curves below threshold were not considered as significant signals (7 and 8).
T63 7033-7577 Sentence denotes Three additional one-step real-time PCR systems were compared with the initially used QuantiTect Virus +Rox Vial kit (QIAGEN) (Table): (i) OneStep RT-PCR kit (QIAGEN; data not shown in the Table because only a limited number of samples were tested in only one run), (ii) LightCycler Multiplex RNA Virus Master (Roche, Mannheim, Germany; data not shown because only a limited number of samples were tested in only one run) and (iii) SuperScript III One-Step RT-PCR system with Platinum TaqDNA Polymerase (Invitrogen, Darmstadt, Germany) (Table).
T64 7578-7740 Sentence denotes Each assay protocol and thermoprofile was adjusted to the recommendations of the manufacturer, but primer and probe concentrations were the same as published [5].
T65 7741-7818 Sentence denotes They all showed comparable results in reduction of unspecific E gene signals.
T66 7819-8180 Sentence denotes Using SuperScript III, the unspecific signals in the E gene screening assay were significantly reduced to 5% (13/257) in tested patient samples and negative extraction and non-template controls (Table), whereas Ct values for positive controls (Wuhan coronavirus 2019 E gene and SARS-CoV Frankfurt 1 genomic RNA [3]) were in the same range in QuantiTect (median:
T67 8181-8193 Sentence denotes 29.1; range:
T68 8194-8240 Sentence denotes 26.8–32.4; n = 85) and Superscript (median Ct:
T69 8241-8253 Sentence denotes 28.1; range:
T70 8254-8280 Sentence denotes 26.4–31.0; n = 30) setups.
T71 8281-8448 Sentence denotes In the following, we decided to switch to the SuperScript III system as recommended by Corman et al. [5], also because of its decreased thermoprofile run-time (Table).
T72 8449-8534 Sentence denotes Moreover, we additionally included a newly launched commercial test kit in our study:
T73 8535-8643 Sentence denotes RealStar SARS-CoV-2 RT-PCR kit 1.0 (Altona, Hamburg, Germany), which did not show unspecific E gene signals.
T74 8644-8752 Sentence denotes A summary of the assay features of the three PCR setups tested and compared in detail is shown in the Table.
T75 8753-8950 Sentence denotes For evaluation of assay performance regarding efficiency, linearity and unspecific signals, we used the SuperScript protocol in a direct comparison of 73 samples with the RealStar SARS-CoV-2 assay.
T76 8951-9183 Sentence denotes The E gene assays showed 97% (71/73) identical results in both assays (60 negative and 11 positive results as well as two divergent results, both with a positive Real Star SARS-CoV-2 assay and a negative SuperScript protocol assay).
T77 9184-9309 Sentence denotes The specific SARS-CoV-2 assays gave in 67 (92%) identical results (60 negative and seven positive) and six divergent results.
T78 9310-9375 Sentence denotes Overall, the RealStar SARS-CoV-2 kit seemed to be more sensitive.
T79 9377-9385 Sentence denotes Workflow
T80 9386-9552 Sentence denotes With increasing sample numbers, it is crucial to implement a new assay into routine laboratory workflows that already exist for diagnostic testing of other pathogens.
T81 9553-9717 Sentence denotes Sample preparation and RNA extraction were carried out at BSL2 level under a safety class 2 cabinet wearing FFP3 filter masks following the WHO recommendations [8].
T82 9718-9875 Sentence denotes Samples arriving at our laboratory before 10:00 h were analysed on the same day and results were reported 6–7 h later to local health authorities (Figure 3).
T83 9876-9918 Sentence denotes Our daily test capacity was ca 80 samples.
T84 9919-10092 Sentence denotes It should be taken into account that well trained and experienced personnel is needed and seasonal pathogens, e.g. influenza and noroviruses, had to be analysed in parallel.
T85 10093-10174 Sentence denotes Overall, we managed to report the results for 97% of samples within the same day.
T86 10175-10288 Sentence denotes Figure 3 Flowchart and routine timeline of the SARS-CoV-2 diagnostic workflow at the LGL, Bavaria, February 2020
T87 10289-10293 Sentence denotes LGL:
T88 10294-10336 Sentence denotes Bavarian Health and Food Safety Authority.
T89 10338-10348 Sentence denotes Discussion
T90 10349-10480 Sentence denotes On 7 January 2020, a novel coronavirus was identified and shortly after, the first sequence of the new strain was published [9,10].
T91 10481-10601 Sentence denotes The main task of public health authorities is to react quickly to emerging pathogens of global threat to prevent spread.
T92 10602-10739 Sentence denotes These responses include containment strategies, which means that close contacts of patients have to be identified and tested immediately.
T93 10740-10968 Sentence denotes Reusken et al. identified the availability of positive control material and primer/probes as well as the lack of skilled personnel and time as the most prominent challenges in the implementation of the new SARS-CoV-2 assay [11].
T94 10969-11065 Sentence denotes National and local public health laboratories play a crucial role in testing emerging pathogens.
T95 11066-11424 Sentence denotes The Public Health Microbiology Laboratory in Bavaria was confronted with SARS-CoV-2-related events very early: once the assays and control materials arrived and the PCR assays were performed for the first time, a large contact investigation around the first German COVID-19 patient (data not shown) was immediately started, with so far more than 700 samples.
T96 11425-11554 Sentence denotes Evaluation and verification of the new assays and testing real samples had to take place simultaneously within a very short time.
T97 11555-11721 Sentence denotes We realised soon that the SARS-CoV E gene assay was more sensitive than the two RdRp gene assays combined with the one-step RT-PCR system available in our laboratory.
T98 11722-11821 Sentence denotes However, our E gene assay showed high background levels hampering a clear evaluation of the assays.
T99 11822-11950 Sentence denotes Each mastermix has its own proprietary composition, which may explain the differences in the performance of a certain PCR assay.
T100 11951-12115 Sentence denotes Using commercial kits with optimised target regions and primer sequences (in the E gene and SARS-CoV-2-specific S gene) ruled out the unspecific signals completely.
T101 12116-12426 Sentence denotes Hence, reasons for the observed unspecific signals may be dimerisation of primers and probes and/or unspecific primer binding and polymerase activity in the targeted region of the E gene, probably also depending on thermal profile and cycler-specific differences, or most likely a combination of these factors.
T102 12427-12695 Sentence denotes Contamination as a reason for unspecific signals was ruled out, as stringent prevention measures were taken, e.g. strict separation of working areas: oligonucleotides and PCR mastermix reagents were handled in one room under a PCR hood with specified laboratory coats.
T103 12696-12762 Sentence denotes Sample preparation and RNA extraction took place in a second room.
T104 12763-12817 Sentence denotes Sample RNA was added in a third room under a PCR hood.
T105 12818-12879 Sentence denotes The synthetic E gene control was added last to the mastermix.
T106 12880-12936 Sentence denotes All reagents were aliquoted and aliquots used once only.
T107 12937-13219 Sentence denotes Contaminations from synthetic E gene present in primer batches upon delivery can be ruled out as well, although only one batch of E gene primers and probes was used with the QuantiTect and Superscript III setup, as only a certain proportion of samples showed the unspecific signals.
T108 13220-13386 Sentence denotes Furthermore, the unspecific signals were significantly reduced in the Superscript III setup, which showed that its sensitivity was comparable to the QuantiTect setup.
T109 13387-13635 Sentence denotes In addition, the initially used E gene primers and probe were separately used as templates with the RealStar kit and no amplification was observed, whereas the corresponding artificial E gene template delivered a clear S-shaped curve with this kit.
T110 13637-13647 Sentence denotes Conclusion
T111 13648-13841 Sentence denotes The SARS-CoV-2 assay from Corman et al. [7] could be used rapidly with the published SuperScript III system, but further optimisation especially of the E gene assay may enhance the sensitivity.
T112 13842-14149 Sentence denotes The RealStar kit outperformed the two other tested one-step PCR systems in sensitivity and by absence of unspecific signals with the used primer set and the RealStar workflow enhanced laboratory efficiency combining two coronavirus assays (lineage B CoV and specific SARS-CoV-2) with an IC in a triplex PCR.
T113 14150-14315 Sentence denotes Overall, fast assay development and publication of protocols by expert laboratories allowed public health laboratories to establish diagnostics quickly and act fast.
T114 14316-14409 Sentence denotes Assay optimisation will further speed up and improve the management of this ongoing outbreak.