References 1. Loeffelholz M. Laboratory diagnosis of emerging human coronavirus infections - the state of the art. Emerg Microbes Infect. Okamaoto K. Jpn J Infect Dis. Suo T. Tahamtan A. Expert Rev Mol Diagn. Chavez V. Sources of pre-analytical, analytical and post-analytical errors in the microbiology laboratory. In: Dasgupta A. Accurate results in the clinical laboratory. A guide to error detection and correction. Elsevier Inc. Kucirka L.
Variation in false negative rate of reverse transcriptase polymerase chain reaction-based SARS-CoV-2 tests by time since exposure. Ann Intern Med. Rahman H. Wernike K. Transbound Emerg Dis. Katz A. False-positive reverse transcriptase polymerase chain reaction screening for SARS-CoV-2 in the setting of urgent head and neck surgery and otolaryngologic emergencies during the pandemic: Clinical implications.
Head Neck. Alternatively, probes can be used in lateral flow assays, whereby cleavage of the probe produces two distinct bands on the flow-strip, indicating a positive result. A single band on the lateral flow strip is representative of a negative result as no cleavage events occurred and the labels are not separated C , bottom. First, primer sets must bind to the target to facilitate initial product amplification, then the guide RNA must detect a specific sequence on this product to facilitate signal generation.
This dual mechanism could help alleviate issues associated with the non-specific primer binding sometimes observed in isothermal amplification. The fact that both amplification and Cas-cleavage can be performed in a single reaction is also appealing, as it reduces the risk of contamination and requires less hands-on time.
Given that already existing isothermal amplification methods can be transitioned to CRISPR-Cas nucleic acid detection, these enhanced assays could be rapidly developed against a host of viruses. It is likely that with the improved specificity that this detection system offers, that its application will continue to grow in the field of virology.
Automated systems based on the amplification of nucleic acids from a sample are, therefore, an extremely useful tool in scenarios where multiple samples require assessment, or where a sample must be tested for the presence of a range of viruses.
Currently, several systems are available for the high-throughput sensitive detection of nucleic acids, examples of which include the BioFire FilmArray, Verigene, Gen-Probe Prodesse and Cepheid systems Babady, ; Van Wesenbeeck et al. Such systems are capable of running multiplexed diagnostic assays, with the number of samples used per run varying depending on the platform. Results turnaround is generally within a day, with some systems capable of rapid testing, producing results within an hour.
In addition to a rapid run time, integrated sample preparation is also available on several systems, reducing hands-on time to as little as several minutes Babady, ; Van Wesenbeeck et al. The offer of contained sample preparation within some automated systems is advantageous when the sample may contain a high-risk viral pathogen Loftis et al.
When large-scale viral detection is required, automated systems may offer an advantage over amplification methods such as isothermal methods due to their amenability to multiplexing and high-throughput analysis.
However, the requirement for specialised equipment may limit the suitability of these automated assays in low resource settings. Nonetheless, the capacity to assay multiple samples simultaneously is highly useful for application in settings where large-scale testing is necessitated. In summation, methods which rely on nucleic acid detection are useful tools for analysing viral infection. They are often very sensitive and specific, ranging in complexity of design from LAMP, which requires at least four primers to function, to RCA which at its minimum only requires one primer.
The higher the number of oligonucleotides which must bind to the target for the assay to proceed generally means a lower rate of non-specific binding and non-target product formation. This is, again, appealing for on-site diagnostics but also for scenarios where time is of the essence and avoiding sample preparation would reduce the time to result.
In addition to qPCR and isothermal techniques, automated DNA amplification-based platforms are an important tool in viral diagnostics as they generally offer quick turnaround times and require limited manipulation of samples.
Another strength of nucleic acid-based detection systems is the speed at which they can be designed when the sequence of the virus of interest is available. Given that viruses can rapidly emerge, the ability to quickly develop assays to detect particularly damaging, pathogenic or transmissible viruses is of major interest.
Therefore, nucleic acid techniques will remain essential in the future of virology. Nonetheless, there are other methods which are important for virus detection. These include methods which employ biological detection agents, such as antibodies, to identify virus-associated proteins.
Immunoassays employ antibodies as the primary means to detect viruses within a sample. This can be useful in situations where the desired outcome is indiscriminate detection of all viral strains, or when the goal is to capture all strains of a virus from a sample.
In contrast, monoclonal antibodies, and certain recombinant forms of antibodies, present singular-epitope specificity, hence, they are monospecific.
Specific antibodies are valued in diagnostics as they offer the opportunity for the targeted detection of distinct regions on the target molecule. In virus diagnosis, this is useful for differentiation between different isolates or genotypes of a single virus, or between similar viral species within a genus Usuda et al. When the targets are highly similar, for example similar strains of a given virus, isolating non-cross-reactive antibodies can be challenging. To overcome this, antibodies can be isolated based on their ability to detect short peptide sequences.
These peptides correspond to unique regions on the target protein, providing the capacity to detect strain-specific regions on an entire protein Geysen et al. When employing this strategy, it is essential to carefully select the target peptide.
Peptides can sometimes adopt conformations which do not reflect the epitope in its native conformation. Therefore, the binding to both the peptide and the native epitope on the target protein must be validated Conroy et al. Overall, targeting highly specific peptides, rather than an entire protein with multiple epitope regions, could result in antibodies which are less likely to cross-react with other viruses or isolates of the viral species. The initial development of pathogen-targeting antibodies requires time to identify a valid disease-specific antigen, isolate suitable antibodies and validate the resulting antibodies and assays.
However, once generated, the production of the antibody proteins is relatively fast. Furthermore, the advent of recombinant antibody technology, whereby antibodies are produced as fragments of the whole antibody protein, has made the generation of antibodies more rapid and cost-effective, as cheaper expression hosts such as yeasts and bacteria can be used in lieu of mammalian cell-culture Jeong et al. Virus-targeting antibodies are used in virology for multiple purposes.
Two major applications are for the detection of virus-associated antigens from samples, and for determining seroprevalence. Seroprevalence assays are essential techniques for identifying patients who have been exposed to a virus Li et al.
However, these assays rely on the identification of anti-virus antibodies generated by the host, whereas this review focuses on the creation, employment and design of specific antibodies to detection pathogen-associated targets.
As such, the following section largely deals with the use of antibodies for identifying virus-associated antigens from samples, with only brief mention to applications for seroprevalence monitoring.
The detection of virus-antigens was initially facilitated by virus propagation through cell culture methods, whereby antibodies are used to detect virus-associated proteins within a cell line, or to perform virus neutralisation assays Leland and Ginocchio, However, as discussed, such cell culture methods are relatively slow when compared to other diagnostic means.
Therefore, much focus is on the development of antibody-based assays capable of detecting the target-virus directly from source through a range of antibody-based assays, termed immunoassays.
There are a number of formats in which the ELISA assay can be performed, namely direct, indirect, competitive and sandwich Figure 6. These formats are also used for most other immunoassays. A In direct detection, the antigen is detected directly by a labelled antibody. Indirect detection is a variation of this format, where the primary antigen-specific antibody is unlabelled, and must be detected by a secondary labelled antibody that selectively binds to the primary antibody.
B In competitive detection, free antigen in the sample competes with immobilised antigen for binding to the antibody which may be directly labelled or may be detected using a secondary labelled antibody. Hence, in competitive immunoassays, the signal is inversely proportional to the concentration of antigen.
C In the sandwich immunoassay format, the antigen is captured by an antibody that reacts with a specific epitope on the antigen. A second labelled antibody is added which reacts with another different epitope on the captured antigen. Here the signal generated is directly proportional to the amount of antigen present.
Direct and indirect formats are similar. Initially, the antigen is coated to the surface of the ELISA well through passive adsorption, or it may be chemically linked. For direct detection, a labelled, anti-target primary antibody is applied to the well, and subsequently detected.
For indirect detection, the primary antibody is unlabelled, and is detected by the addition of a labelled, secondary antibody. These formats are useful for measuring an antibody response against a given antigen. However, there are some drawbacks to direct and indirect formats when transitioning to detecting viruses in crude samples such as blood, stool or other tissues.
Most biological samples will contain a combination of target proteins and other proteins. These other proteins compete for adsorption to the assay well surface, meaning that the target-antigen may not be accurately represented in the sample, making quantitation challenging He, To overcome this, formats such as competitive and sandwich are used. Competitive assays are effective in the detection of viral-specific proteins from a given source. Competitive ELISA first involves the immobilisation of the target antigen to an ELISA well, followed by the simultaneous application of the antibody and the suspect sample to the same well.
Target-antigen in the sample competes for antibody-binding, leaving less antibody available for binding to the immobilised antigen. The sample is washed away, and any remaining bound antibody is detected with a labelled, secondary antibody. Therefore, in this assay format, the signal is inversely proportional to the amount of analyte in the sample.
Competitive analysis is widely used for the detection of small antigens, where the binding of multiple antibodies may not be permitted He, This assay format is useful when considering the rapid-development of viral-detection assays as it only requires one target-specific antibody. However, the use of only one antibody also has consequences in the form of reduced assay sensitivity and specificity if any cross-reactivity with the sample matrix occurs. The employment of a sandwich formats overcomes most issues associated with cross-reactivity as it employs two antibodies, both specific for different regions epitopes on the target molecule.
The first of these is immobilised to the well surface. Thereafter, the sample is applied, and specific antigen is captured by the immobilised antibody. Unbound entities are washed away, and the second antibody is applied. This second antibody can be labelled directly or detected with a labelled-secondary antibody. This format offers high specificity as it requires the binding of two antibodies to produce a signal.
This could be helpful for virus detection where there is a risk of cross-reactivity occurring between similar strains of virus. Sandwich assays are less prone to cross-reactivity. Even if one of the antibodies in the pair has some cross-reactivity with the sample matrix, the likelihood is that the same cross-reactivity will not be observed with the alternate binding antibody. This makes this assay suitable for the detection of viruses directly from their sources, such as serum or tissue samples, where the matrix can be very complex Jansen van Vuren and Paweska, ; Zhang et al.
A drawback to the sandwich assays is the complexity of design. Both antibodies need to bind, unhindered by one another, to the target molecule. Finding a suitable binding pair can be difficult, and thorough assay validation is required to ensure no non-specific interaction occurs between the antibodies within the assay. However, the use of antibody-discovery technology, such as phage display, has led to the development of methods which facilitate the identification of binding pairs of antibodies in a rapid manner, negating the time-consuming screening associated with finding two separate binding pairs Gorman et al.
Alternatively, the previously described strategy of peptide-targeting can be employed to design antibodies which can detect distinct regions on the whole antigen molecule, facilitating sandwich detection.
ELISA continues to remain a staple platform in virus detection due to its sensitivity and robustness. However, there are drawbacks to its use. These include the risk of cross-reactivity of antibodies to other co-infecting viruses, resulting in false positives or inaccurate quantification.
This highlights the need to fully validate virus-assays prior to use. In contrast to a potential for false positives, ELISA, and other immunoassays, may also be at risk of presenting false negative results.
This generally occurs when testing is performed at the early window of infection. At this stage, the quantities of viral antigen may be present at low concentrations, challenging the sensitivity of the ELISA format, potentially leading to erroneous results Tillmann, ; Alexander, This limits the capacity for rapid testing and results turnaround. However, the multi-well layout of ELISA plates facilitates multiple simultaneous tests, which is ideal for scenarios where high volume testing is required, such as epidemic or pandemic scenarios.
In order to improve this turnaround time other antibody-based methods are suggested for virus detection. Blotting techniques generally involve antigen detection on the surface of a membrane. The dot blot, or slot blot, is a technique which can be used for the detection of viral antigens from a sample Li et al.
To do this, usually the suspect sample is blotted onto a membrane, allowed to dry and the membrane is then probed with an anti-virus antibody. This method is cost effective and offers advantages such as the requirement for a small sample volume, the ability to detect virus directly from the sample and the fact that blotted membranes can be stored for a number of days before testing, facilitating the testing of numerous membranes simultaneously.
However, the results of dot-blots are mostly qualitative, requiring further instrumentation to perform quantitive analysis which may increase costs. These involve pressing of the infected tissue onto the membrane to facilitate virus antigen immobilisation. Blotting techniques are useful for cheap detection of multiple samples, as many samples can be blotted on the same membrane. However, the time required to produce results is a considerable drawback, as the turnaround may take several hours.
Schematic representation of the detection of viral antigens through dot blot or tissue blot. The mixture of antigens within the sample is blotted onto the membrane and thereby immobilised. The target antigens are detected using target-specific labelled antibodies.
The assays are typically formatted in either sandwich or competitive immunoassay formats Sajid et al. The assay result is usually read by way of a colour change at a test line. In sandwich format, a colour change at this line corresponds to a positive result, whereas in competitive format no colour change is indicative of a positive result. Control lines are incorporated into both assays to ensure test validity.
The colour change is facilitated by the labelling of antibodies with various molecules. Labels such as gold nanoparticles, coloured latex beads, carbon nanoparticles and magnetic particles facilitate one-step results where a colour change can be observed with the naked eye, facilitating rapid diagnosis.
Other labels such as fluorophores, quantum dots and enzymatic labels can also be employed, however these may require additional equipment or steps to demonstrate results which may be qualitative or quantitative Posthuma-Trumpie et al.
One primary advantage is that LFIAs have a turnaround time of minutes, rather than hours. Further to this, performing a LFIA requires minimal sample clean up, having been proven to efficiently detect viral antigens from crude samples, including viral transport media and blood Ryu et al. Assays can also be performed using saliva samples providing a non-invasive and easy-to-acquire source for viral testing Yoon et al. LFIAs are also cost effective, requiring minimal sample volumes to prepare and facilitating diagnosis with the naked eye.
However, this also comes with the drawback of only offering qualitative results. To achieve quantitative results, further instrumentation is required Mak et al. Another advantage of LFIA is their ease-of-use, which makes the technology accessible to a wide range of users. This can be helpful in scenarios where a high volume of testing is required, as non-specialists can be quickly trained to performs assays. Overall, immunoassays in viral detection play an essential role.
While they may not always offer the same sensitivity as nucleic-acid detection methods, particularly at early stages of infection, they come with the advantage of a reduced cost, reduced complexity and higher utility for use by untrained personnel. These factors are exemplified by the LFIA which can be produced cheaply, has no need for additional equipment and requires little to no training to use. Table 1 provides a comparison between the various nucleic acid detection-based and immunoassay-based methods.
The specificity of antibodies in immunoassays is one of the primary challenges. This is exacerbated in virology by the presence of multiple strains of a single virus, or by similar viruses within a genus. Recombinant antibody technology facilitates rapid re-engineering of antibody characteristics such as specificity and offers a means to alter antibodies for reduced cross-reactivity Ducancel and Muller, ; Ma et al.
This could aid in overcoming any specificity-related obstacles. Antibody discovery using recombinant technology also has a faster turnaround when compared to methods such as hybridoma technology Basu et al. Therefore, recombinant technology should be considered an essential tool in the development of future viral diagnostics assays for both existing and emergent viral strains.
Summary of the advantages, disadvantages and example applications off nucleic acid-based and immunoassay-based approaches to virus detection. While molecular and antibody-based techniques will likely continue to dominate the field of virology, their use should be complemented by engaging with newer technologies, such as next generation sequencing NGS platforms. The employment of NGS benefits virology in multiple ways. Firstly, it can act as a stand-alone method for viral diagnostics, if required.
This, therefore, provides a powerful tool to diagnose viruses, and would be particularly useful in scenarios where patients present with a disease and the causative-pathogen is unidentified Barzon et al. Therefore, while currently established methods may be more rapid in diagnosing known viruses, NGS should be a promising way to reduce time-to-discovery for novel, or unexpected, viral pathogens.
Instances of NGS implementation providing a diagnosis to a previously unknown virus, or identifying unexpected viruses in samples, are reported, highlighting its usefulness Palacios et al. Similarly, monitoring of viral levels in a population could also be facilitated by NGS, with particular note to monitoring of emergent or unexpected viral strains.
Passive sampling of airborne viruses in crowded areas such as transit networks, schools, day-cares and emergency rooms could be used in conjunction with NGS to assess the viral burden and patterns in particular settings Choi et al. In a similar manner, viral vectors or transmitters, such as mosquitoes or bats, can be sampled passively, and routinely tested, to allow surveillance of virus prevalence in the harbouring population Eiras et al.
This could be helpful for predicting disease outbreaks or novel virus emergence, Another benefit of NGS in virology is the promise of expanding viral genome-sequencing data. The paucity of information with regards to viral sequences can make experimental design difficult, for example, designing broad-spectrum amplification primers or identifying conserved antigens can be challenging when not all viral strains are known Barzon et al.
The capability to sequence many viruses from a population, and hence achieve a more rounded picture of the variation in those viruses, could aid not only in the surveillance of emerging viruses, but in providing additional information to researchers looking to develop novel assays Rosario and Breitbart, ; Radford et al.
While NGS is a promising technology as a tool for virus discovery, there are challenges to overcome. These include the high costs, the need for both trained staff and bioinformaticians, a potential for poor sensitivity in low viral-load infections and a lack of universal sequence data-analysis tools Beerenwinkel et al.
Once addressed, NGS could work in tandem with established techniques to provide a means to identify, characterise and diagnose viral infection. Presently, no single method meets every demand of virus detection. Nucleic acid-based detection is highly sensitive and specific. Its drawbacks include the fact that often trained staff are required, associated costs can be high, and carry-over contamination can cause issues in result analysis.
In contrast, antibody-based detection is generally cheaper, more robust, and more accessible to untrained users. However, immunoassays do not typically achieve the same sensitivity as nucleic acid detection methods.
As such, the decision of whether to employ nucleic acid detection or immunoassays methods will largely be dictated by the requirements of the assay. As NGS becomes more accessible, both nucleic-acid detection and immunoassay methods will benefit from its application.
As the use of NGS expands, so too will the number of assays to facilitate the detection of viruses, driven by an increased knowledge of the diversity of viral genomes and their roles. AC authored the initial review draft.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. National Center for Biotechnology Information , U. Journal List Front Mol Biosci v. Front Mol Biosci. Published online Apr Cassedy , 1 A.
Parle-McDermott , 1 and R. Author information Article notes Copyright and License information Disclaimer. Corresponding author. O'Kennedy, moc. This article was submitted to Molecular Diagnostics and Therapeutics, a section of the journal Frontiers in Molecular Biosciences. Received Dec 3; Accepted Feb 1. The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice.
No use, distribution or reproduction is permitted which does not comply with these terms. This article has been cited by other articles in PMC. Abstract Viruses are ubiquitous in the environment. Keywords: immunoassay, isothermal amplification, next generation sequencing, virus, nucleic-acid detection, sampling. Introduction Viruses are ubiquitous entities which rely on host organisms to replicate. Indirect Detection Indirect detection methods involve the propagation of virus particles via their introduction to a suitable host cell line.
Enhanced Virus Isolation Methods Centrifuge-enhanced shell-vial techniques or well plates, termed cluster plates, facilitate rapid adsorption of the virus into the host cell line by applying low level centrifugation, greatly reducing the length of time required for inoculation of the cell line.
Diagnostic Methods in Virus Isolation Once growing in cell-culture, there are two categories of methods by which viruses are diagnosed. Open in a separate window. Direct Detection Direct detection methods negate the need for virus propagation. Nucleic Acid-Based Detection Methods A key element of nucleic acid-based detection is polymerase chain reaction PCR which utilises multiple stepwise temperature cycles, and a polymerase, to amplify DNA strands Mullis et al.
Immunoassay-Based Viral Diagnostics Immunoassays employ antibodies as the primary means to detect viruses within a sample. Blotting Techniques Blotting techniques generally involve antigen detection on the surface of a membrane. TABLE 1 Summary of the advantages, disadvantages and example applications off nucleic acid-based and immunoassay-based approaches to virus detection.
Method Advantages Disadvantages Examples Refs. Future Directions While molecular and antibody-based techniques will likely continue to dominate the field of virology, their use should be complemented by engaging with newer technologies, such as next generation sequencing NGS platforms.
Conclusion Presently, no single method meets every demand of virus detection. Author Contributions AC authored the initial review draft. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. References Adams I. Next-generation sequencing and metagenomic analysis: a universal diagnostic tool in plant virology. Plant Pathol. Human immunodeficiency virus diagnostic testing: 30 Years of evolution.
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Using both the methods, sample P1 was found to be negative; besides, five of the six NP samples and four of the four saliva samples had lower N1 and N2 Ct values in the Chelex method as compared to the RNA extraction method Figure 3 C. Thus, the Chelex method may offer better sensitivities by using the procedure discussed here. In addition, the Chelex method allowed sample processing without a Biosafety Cabinet hood as the samples were inactivated before the tube was opened. We then determined whether viral and cellular RNAs were stable over time.
Samples were stored at room temperature, then heat treated in the presence of Chelex, and assayed at different time points as indicated Figure 4. A similar stability experiment was performed for virions prepared in saliva samples. The viral RNAs were stable in saliva before heat treatment, as a higher amount of viral RNAs were detected after storage at room temperature, possibly because interfering agents in saliva degrade during storage Figure 5 A.
This is likely due to the enrichment arising from the lower volume of RNA eluate as compared to the input sample volume. When the samples were tested side by side Figure 3 B , the sensitivities of the Chelex method and RNA extraction method were comparable. Un-processed samples may lead to more RNA degradation during sample collection and storage as compared to being stored in the presence of Chelex.
In addition, the lower volume used for sample collection as shown in Figure 3 C could increase RNA detection due to less absorbance of RNA to the tubes. The potential benefits of salivary testing include lower cost no swab , reduced variability, and improved patient acceptance over traditional NP swab Barat et al. Thus, the Chelex method may provide a more sensitive point-of-care method for RNA diagnostics by reducing false-negative results.
The sheer volume of preprints and publications in a short period of time illustrates the urgent need and hope to increase the testing capacity employing the RNA-extraction-free approach. The utility of this RNA isolation method for both NP and saliva samples would increase the number of people tested in the same time frame as the current method.
In addition to improved sensitivity, this method offers a number of additional advantages compared to the current gold standard clinical laboratory testing, including improved cost, reduced sample processing time and complexity, and enhanced workflow safety.
The Chelex-based RNA preparation method—amenable for high-throughput processing—is expected to shorten diagnostic testing time by omitting the RNA extraction step and omitting the chemical disinfection of patient samples. This method utilizes a heat inactivation step that minimizes viral RNA loss and inactivates the virus before the tube is opened, thereby limiting the exposure of laboratory personnel to a live virus.
Therefore, we fully expect that this method will facilitate broader availability and testing capacity not only for COVID but also for other infectious pathogens. Because of the observed stability of SARS-CoV-2 RNA in collected samples at room temperature, this method should also improve access to COVID testing in resource-scarce regions of the world, by improving the RNA stability, reducing the cost of collection kits and diagnostic reagents, and eliminating the requirement of refrigeration, biosafety cabinet, and the storage of RNA extraction kits.
Because the Chelex method also allowed cellular RNA detection, we expect that the method could find wide use in both clinical and research laboratories. DNA present in the solution is not expected to interfere with many applications because RT-qPCR can be performed using exon-spanning primers, and poly A selection is often an integrated step in RNAseq.
This helps to alleviate the concerns of choosing a proper house-keeping gene during RNA expression analysis Panina et al. If DNA needs to be removed before downstream application, Dnase I treatment may be performed using commercially available Dnase I kits.
In summary, we robustly demonstrate improvements in COVID viral testing workflow using synthetic and real-world samples employing the Chelex-based extraction-free workflow. This methodology has clear benefits such as dramatic improvement of sensitivity, cost, and time saving for clinical laboratory testing.
In addition, this method exhibits improved safety characteristics. Finally, this method can be easily adapted by both clinical and research laboratories and could be a standard of care for nucleic acid testing and transport in the near future.
One limitation of the current study was the low-level contamination observed in the RT-ddPCR assay, where one or two positive droplets were observed in the no-template control reactions. Due to background contamination, we used 1. Another limitation of the current study is that the ATCC heat-inactivated virions were used in the method development and room temperature stability assays. However, results in Figure 3 C showed that Chelex-TE buffer may have preserved the virion RNA better because five of the six positive patient samples had 1.
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Robert Hufnagel robert.
Participants of age 18 and older provided samples for individual data points. We do not anticipate the sex of the participants to have any influence on the results. We used four types of samples for assay development and validation: i simulated samples containing the heat-inactivated SARS-CoV-2 virions obtained from ATCC VRHK, concentration measured by ddPCR at ATCC ; ii positive patient saliva samples diluted into negative saliva samples; iii historical patient samples including nasopharyngeal swab and whole unstimulated saliva; and iv prospective paired nasopharyngeal and saliva samples swabs.
Swabs were then inserted into VTM. In parallel, NP samples were placed in 0. The 0. The positive signal for N1 or N2 alone was defined as an indeterminate result. Molecular biology grade water cat , TE pH 8. Individual Ct values were plotted in dot plots. Data in this study were descriptive and no statistical analyses were employed. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services nor does mention of trade names, commercial products, or organizations imply endorsement by the U.
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