LitCovid-sentences  

PMC:7574920 / 32591-42876 JSONTXT 9 Projects

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Id Subject Object Predicate Lexical cue
T249 0-10 Sentence denotes DISCUSSION
T250 11-117 Sentence denotes Here, we evaluated the use and suitability of the RT-LAMP assay for the detection of SARS-CoV-2 infection.
T251 118-256 Sentence denotes We also developed LAMP-sequencing as a fully scalable alternative to colorimetric or fluorometric analysis of DNA amplification reactions.
T252 257-468 Sentence denotes Our results indicate that whereas the RT-LAMP assay using the N-A primer set is not sensitive enough to replace RT-qPCR in all applications, it does hold promise as a method for testing large numbers of samples.
T253 469-632 Sentence denotes We tested the RT-LAMP primer sets suggested by Zhang et al. (11) and found that the N-A primer set for the N gene worked better than the 1a-A primer set for ORF1a.
T254 633-901 Sentence denotes For samples with a CT ≤ 30 as measured by RT-qPCR with E-Sarbeco primers, we found overall satisfactory sensitivity and specificity values for SARS-CoV-2 RNA detection by the RT-LAMP assay using RNA samples isolated from pharyngeal swab specimens (Fig. 3 and Table 1).
T255 902-970 Sentence denotes For samples with CT > 30, the RT-LAMP assay was much less sensitive.
T256 971-1088 Sentence denotes However, there is debate about which CT value for a positive RT-qPCR result should be considered clinically relevant.
T257 1089-1187 Sentence denotes Vogels et al. (16) indicate that a CT value above 36 corresponds to less than 10 molecules of RNA.
T258 1188-1354 Sentence denotes On the basis of our data, we conclude that the colorimetric RT-LAMP assay would be suitable for identifying individuals with a high or moderate SARS-CoV-2 viral load.
T259 1355-1614 Sentence denotes On the other hand, for those with a low viral load (at the onset of illness or during later stages of the disease), the sensitivity of the RT-LAMP assay, in its current implementation using the N-A primer set, is insufficient to detect a SARS-CoV-2 infection.
T260 1615-1876 Sentence denotes A number of other LAMP primer sets have been proposed and initially tested (21, 27, 28), showing that optimized primers and the use of combinations of primer sets hold promise to further increase the sensitivity of the RT-LAMP assay for detecting viral genomes.
T261 1877-1967 Sentence denotes Furthermore, alternative sample types, e.g., sputum or stool (29), might be more reliable.
T262 1968-2194 Sentence denotes One promising lead for future applications is the exploration of the hot swab–to–RT-LAMP assay using saliva specimens, although the relative sensitivity compared to using pharyngeal swab specimens is currently unclear (30–33).
T263 2195-2310 Sentence denotes Compatibility of the RT-LAMP assay with direct saliva specimens has been shown using spike-in experiments (22, 34).
T264 2311-2458 Sentence denotes Although faster and more convenient, the direct swab–to–RT-LAMP assay was less sensitive and less robust than the RT-LAMP assay using isolated RNA.
T265 2459-2721 Sentence denotes To increase robustness, various treatments of crude swab samples have been described previously [reviewed in (35)], many of which require additional processing of the samples, for example, by pipetting or by adding proteinase K to degrade contaminating proteins.
T266 2722-2939 Sentence denotes Rabe and Cepko (22) have suggested using cheap silica preparations and new sample inactivation protocols to enrich the RNA before the RT-LAMP assay, but this would complicate the simple swab–to–RT-LAMP assay workflow.
T267 2940-3195 Sentence denotes Last, our analysis found that a short heat treatment of 5 min at 95°C, which poses minimal additional handling steps, did not destroy the RNA but rather stabilized it and this improved the sensitivity and specificity of the swab–to–RT-LAMP assay (Fig. 5).
T268 3196-3341 Sentence denotes The heat likely helped to homogenize the sample, to inactivate ribonucleases (RNAses), and to break up the viral capsid to release the viral RNA.
T269 3342-3539 Sentence denotes Overall, our data demonstrate the feasibility of using a swab–to–RT-LAMP test and suggest applications especially in scenarios where RNA isolation is not available, e.g., in resource-poor settings.
T270 3540-3719 Sentence denotes In such cases, the hot swab–to–RT-LAMP assay seems a good option given that the direct swab–to–RT-LAMP assay yields a number of false positives due to spurious amplification (14).
T271 3720-3924 Sentence denotes Although spike-in experiments with IVT RNA can be informative, we have experienced clear differences when comparing such experiments to those using clinical RNA samples isolated from swab specimens (figs.
T272 3925-3954 Sentence denotes S6 and S7, and data file S1).
T273 3955-4130 Sentence denotes We therefore recommend validating any new proposed rapid SARS-CoV-2 diagnostic test using “real-life” clinical samples including a large fraction of negative clinical samples.
T274 4131-4315 Sentence denotes To overcome the problem of spurious amplification, an expanded oligonucleotide set that incorporates sequence-specific probes (34) or a CRISPR/Cas12a–based approach (36) could be used.
T275 4316-4413 Sentence denotes However, these applications have yet to be tested with large numbers of diverse clinical samples.
T276 4414-4482 Sentence denotes There are several differences between the RT-LAMP assay and RT-qPCR.
T277 4483-4712 Sentence denotes First, RT-qPCR requires a thermocycler to conduct the DNA amplification reaction, which is an expensive instrument, whereas isothermal incubation of RT-LAMP reactions can be conducted using a simple water bath or a heating block.
T278 4713-4787 Sentence denotes This makes the RT-LAMP assay more amenable for point-of-care applications.
T279 4788-4905 Sentence denotes Second, the reagents for the RT-LAMP assay are different from the ones used for RT-qPCR and are supplier independent.
T280 4906-5076 Sentence denotes According to the supplier of the RT-LAMP reagents used in this study (New England Biolabs), production of RT-LAMP reagents can be easily ramped up to satisfy high demand.
T281 5077-5251 Sentence denotes Third, the RT-LAMP assay, when combined with LAMP-sequencing, is suitable for analyzing large numbers of RT-LAMP reactions owing to the fully scalable DNA barcoding strategy.
T282 5252-5389 Sentence denotes In contrast, there are several hurdles to scaling up RT-qPCR assays, the major hurdle being the need for a large number of thermocyclers.
T283 5390-5493 Sentence denotes The RT-LAMP assay overcomes this problem and therefore will be a more scalable method for mass testing.
T284 5495-5560 Sentence denotes Application of RT-LAMP and LAMP-sequencing for SARS-CoV-2 testing
T285 5561-5668 Sentence denotes With its good sensitivity for samples up to CT ≈ 30, the colorimetric RT-LAMP assay has several advantages:
T286 5669-5740 Sentence denotes It is fast, inexpensive, and it can be evaluated without any equipment.
T287 5741-5989 Sentence denotes RT-LAMP reactions also appear to be less sensitive to contaminants in the samples than RT-qPCR, but care has to be taken that the samples used do not alter the pH as the colorimetric RT-LAMP assay is performed under conditions of weak pH buffering.
T288 5990-6154 Sentence denotes Some clinical samples contain contaminants that can lead to acidification of the reaction independent of the presence of a template RNA if too much sample is added.
T289 6155-6432 Sentence denotes Diagnostic RT-qPCR tests usually include a technical internal control, i.e., another RNA species, which is spiked into all samples and which is detected independent of the gene of interest to safeguard against the possibility of a general reaction failure within a sample tube.
T290 6433-6506 Sentence denotes It would be desirable to have a similar precaution for the RT-LAMP assay.
T291 6507-6635 Sentence denotes A multiplexed fluorescence readout might provide this (34) but comes at the expense of the simplicity of a colorimetric readout.
T292 6636-6930 Sentence denotes Our particular implementation of deep sequencing to analyze many RT-LAMP reactions simultaneously uses two sets of barcoded primers and is fully scalable so that, in one sequencing run, many thousands of LAMP reactions can be quantitatively analyzed for the presence of viral genomic sequences.
T293 6931-7117 Sentence denotes Although we used Illumina dye sequencing, more scalable sequencing technologies, such as Oxford Nanopore Technologies sequencing, could be used for amplicon sequencing and counting (37).
T294 7118-7262 Sentence denotes The workflow shown here uses LAMP-sequencing as a validation and backup procedure to double check the results of the colorimetric RT-LAMP assay.
T295 7263-7498 Sentence denotes However, LAMP-sequencing could also facilitate scale-up of the workflow for direct analysis of many thousands of samples in an efficient manner, provided that an infrastructure is established that allows the collection of such samples.
T296 7499-7606 Sentence denotes Thus, LAMP-sequencing could become an important part of workflows for routine testing of large populations.
T297 7607-7818 Sentence denotes Schmid-Burgk et al. (38) proposed decentralized RT-LAMP assays using combinatorial primer barcoding and centralized mass analysis of RT-LAMP products by next-generation sequencing as a means to scale-up testing.
T298 7819-8044 Sentence denotes Although this poses additional challenges in generating the individualized RT-LAMP assay reagents, it would simplify sample handling on the analytical side and it can be easily combined with the barcoding strategy shown here.
T299 8045-8088 Sentence denotes There are several limitations to our study.
T300 8089-8199 Sentence denotes We used surplus RNA sample material from a diagnostic laboratory rather than newly collected clinical samples.
T301 8200-8303 Sentence denotes The criteria for testing individuals may have influenced cohort characteristics and hence our findings.
T302 8304-8475 Sentence denotes It is not clear yet how well viral load as indicated by CT values from RT-qPCR assays informs about the degree of infectivity of an individual with a SARS-CoV-2 infection.
T303 8476-8687 Sentence denotes Therefore, we cannot say how our findings on the sensitivity of the RT-LAMP assay in comparison to RT-qPCR would translate into sensitivity for detecting infectious individuals who are shedding SARS-CoV-2 virus.
T304 8688-8888 Sentence denotes Moreover, the measured viral load does not indicate the course of a SARS-CoV-2 infection, as even individuals with a very low measured viral load can still develop severe symptoms of COVID-19 disease.
T305 8889-9088 Sentence denotes This may be, in part, because the viral load in a clinical sample taken from a specific site such as the pharynx is not representative of the overall viral burden that an infected individual carries.
T306 9089-9191 Sentence denotes We used LAMP-sequencing to validate the RT-LAMP assay results and did not use it as a diagnostic tool.
T307 9192-9369 Sentence denotes LAMP-sequencing is dependent on the sensitivity of the RT-LAMP reaction as it cannot detect false negative results caused by a failure of the RT-LAMP assay to amplify viral RNA.
T308 9370-9491 Sentence denotes Also, reagents such as the primer sets for the RT-LAMP assay may be subject to production-dependent quality fluctuations.
T309 9492-9605 Sentence denotes Therefore, all reagents must be precisely validated (batch control) before using an RT-LAMP assay diagnostically.
T310 9606-9720 Sentence denotes Application of the RT-LAMP assay has great potential, even more so as more sensitive primer sets become available.
T311 9721-9861 Sentence denotes The RT-LAMP assay and LAMP-sequencing could offer scalable testing that would be difficult to achieve with conventional RT-qPCR–based tests.
T312 9862-10060 Sentence denotes For example, the RT-LAMP assay could be used for regular testing of a whole workforce or in sentinel testing, ideally combined with simplified sample collection, e.g., in the form of saliva samples.
T313 10061-10285 Sentence denotes The RT-LAMP assay and LAMP-sequencing extend the range of available test methods and complement individual tests and pooled tests based on RT-qPCR (39) with a faster, simpler, and potentially more cost-effective test method.