Single-tube collection and nucleic acid analysis of clinical samples for SARS-CoV-2 saliva testing.

The SARS-CoV-2 pandemic has brought to light the need for expedient diagnostic testing. Cost and availability of large-scale testing capacity has led to a lag in turnaround time and hindered contact tracing efforts, resulting in a further spread of SARS-CoV-2. To increase the speed and frequency of testing, we developed a cost-effective single-tube approach for collection, denaturation, and analysis of clinical samples. The approach utilizes 1 µL microbiological inoculation loops to collect saliva, sodium dodecyl sulfate (SDS) to inactivate and release viral genomic RNA, and a diagnostic reaction mix containing polysorbate 80 (Tween 80). In the same tube, the SDS-denatured clinical samples are introduced to the mixtures containing all components for nucleic acids detection and Tween 80 micelles to absorb the SDS and allow enzymatic reactions to proceed, obviating the need for further handling of the samples. The samples can be collected by the tested individuals, further decreasing the need for trained personnel to administer the test. We validated this single-tube sample-to-assay method with reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) and discovered little-to-no difference between Tween- and SDS-containing reaction mixtures, compared to control reactions. This approach reduces the logistical burden of traditional large-scale testing and provides a method of deployable point-of-care diagnostics to increase testing frequency.

Portable real-time colorimetric LAMP-device for rapid quantitative detection of nucleic acids in crude samples.

Loop-mediated isothermal amplification is known for its high sensitivity, specificity and tolerance to inhibiting-substances. In this work, we developed a device for performing real-time colorimetric LAMP combining the accuracy of lab-based quantitative analysis with the simplicity of point-of-care testing. The device innovation lies on the use of a plastic tube anchored vertically on a hot surface while the side walls are exposed to a mini camera able to take snapshots of the colour change in real time during LAMP amplification. Competitive features are the rapid analysis (< 30 min), quantification over 9 log-units, crude sample-compatibility (saliva, tissue, swabs), low detection limit (< 5 copies/reaction), smartphone-operation, fast prototyping (3D-printing) and ability to select the dye of interest (Phenol red, HNB). The device's clinical utility is demonstrated in cancer mutations-analysis during the detection of 0.01% of BRAF-V600E-to-wild-type molecules from tissue samples and COVID-19 testing with 97% (Ct < 36.8) and 98% (Ct < 30) sensitivity when using extracted RNA and nasopharyngeal-swabs, respectively. The device high technology-readiness-level makes it a suitable platform for performing any colorimetric LAMP assay; moreover, its simple and inexpensive fabrication holds promise for fast deployment and application in global diagnostics.

Convective polymerase chain reaction in standard microtubes.

Polymerase chain reaction (PCR) is the most widely used method for nucleic acids amplification. To date, a huge number of versatile PCR techniques have been developed. One of the relevant goals is to shorten PCR duration, which can be achieved in several ways. Here, we report on the results regarding nucleic acids amplification by convective PCR (cPCR) in standard 0.2 ml polypropylene microtubes. The following conditions were found to be optimal for such amplification: 1) 70 µl reaction volume, 2) the supply of external temperature 145°Ð¡ for the denaturation zone and 0°Ð¡ for the annealing zone, 3) ∼30° inclination of the microtube main axis, 4) the use of nearby primers, and 5) duration of the reaction 15-20 min. At these conditions, the amplification products are accumulated in an amount sufficient to be registered by gel electrophoresis, and high sensitivity of the reaction comparable to that of conventional PCR is achieved. cPCR provided the reliable detection of SARS-CoV-2 coronavirus RNA isolated from nasopharyngeal swabs of COVID-19 patients.

Automatic system for high-throughput and high-sensitivity diagnosis of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had severe consequences for health and the global economy. To control the transmission, there is an urgent demand for early diagnosis and treatment in the general population. In the present study, an automatic system for SARS-CoV-2 diagnosis is designed and built to deliver high specification, high sensitivity, and high throughput with minimal workforce involvement. The system, set up with cross-priming amplification (CPA) rather than conventional reverse transcription-polymerase chain reaction (RT-PCR), was evaluated using more than 1000 real-world samples for direct comparison. This fully automated robotic system performed SARS-CoV-2 nucleic acid-based diagnosis with 192 samples in under 180 min at 100 copies per reaction in a "specimen in data out" manner. This throughput translates to a daily screening capacity of 800-1000 in an assembly-line manner with limited workforce involvement. The sensitivity of this device could be further improved using a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based assay, which opens the door to mixed samples, potentially include SARS-CoV-2 variants screening in extensively scaled testing for fighting COVID-19.

3D-printed swab with cover for precision diagnosis.

The collection capacity of common nasopharyngeal swabs and irregularities of medical personnel limit the accuracy of PCR testing. This study describes a newly designed 3D-printed swab that is combined with a 3D-printed cover to prevent the extraction of undesired nasal secretions. This swab improved the accuracy of PCR test results. The results of a series of experiments showed that, because of the mucus extraction effect, 3D-printed swabs can replace ordinary cotton swabs. The crisis of the worldwide medical supply shortage can be ameliorated to a certain extent by applying 3D printing technology.

Loop-mediated isothermal amplification (LAMP): An effective molecular point-of-care technique for the rapid diagnosis of coronavirus SARS-CoV-2.

The novel coronavirus disease-2019 (Covid-19) public health emergency has caused enormous loss around the world. This pandemic is a concrete example of the existing gap between availability of advanced diagnostics and current need for cost-effective methodology. The advent of the loop-mediated isothermal amplification (LAMP) assay provided an innovative tool for establishing a rapid diagnostic technique based on the molecular amplification of pathogen RNA or DNA. In this review, we explore the applications, diagnostic effectiveness of LAMP test for molecular diagnosis and surveillance of severe acute respiratory syndrome coronavirus 2. Our results show that LAMP can be considered as an effective point-of-care test for the diagnosis of Covid-19 in endemic areas, especially for low- and middle-income countries.

WHotLAMP: A simple, inexpensive, and sensitive molecular test for the detection of SARS-CoV-2 in saliva.

Despite the development of effective vaccines against SARS-CoV-2, epidemiological control of the virus is still challenging due to slow vaccine rollouts, incomplete vaccine protection to current and emerging variants, and unwillingness to get vaccinated. Therefore, frequent testing of individuals to identify early SARS-CoV-2 infections, contact-tracing and isolation strategies remain crucial to mitigate viral spread. Here, we describe WHotLAMP, a rapid molecular test to detect SARS-CoV-2 in saliva. WHotLAMP is simple to use, highly sensitive (~4 viral particles per microliter of saliva) and specific, as well as inexpensive, making it ideal for frequent screening. Moreover, WHotLAMP does not require toxic chemicals or specialized equipment and thus can be performed in point-of-care settings, and may also be adapted for resource-limited environments or home use. While applied here to SARS-CoV-2, WHotLAMP can be modified to detect other pathogens, making it adaptable for other diagnostic assays, including for use in future outbreaks.

Regenerated silica-based RNA purification columns to address the short supply of RNA purification kits for COVID-19 diagnosis.

BACKGROUND: RT-qPCR technique is the current world-wide method used for the early detection of SARS-CoV2 RNA in the suspected clinical samples. Viral RNA extraction is the key pre-analytical step for SARS-CoV2 detection which often achieved using commercial RNA-extraction kits. However, due to the COVID-19 pandemic, bulk production and the supply chains for the commercial RNA-extraction kit have been seriously compromised. The shortage of commercial RNA-extraction kit is even more acute in developing country. Furthermore, use of one-off design RNA-columns can generate plastic wastes that have an environmental pollution effect. METHODS AND RESULTS: To address these issues, in this study, we used warm alkaline solution containing Triton X-100 for the complete removal of the residual SARS-CoV2 RNA from the used RNA-binding silica column. Columns regenerated using the alkaline solution have the viral RNA purification capability that is comparable to the fresh silica columns. We also demonstrated that RNA-binding silica columns can be regenerated and reused for a minimum of five-times. CONCLUSIONS: Therefore, the use of the RNA-column regeneration method may benefits several SARS-CoV2 diagnostic laboratories throughout the world by cutting down the requirement of commercial RNA-purification column.

Implementation of an open-source robotic platform for SARS-CoV-2 testing by real-time RT-PCR.

The current global pandemic due to the SARS-CoV-2 has pushed the limits of global health systems across all aspects of clinical care, including laboratory diagnostics. Supply chain disruptions and rapidly-shifting markets have resulted in flash-scarcity of commercial laboratory reagents; this has motivated health care providers to search for alternative workflows to cope with the international increase in demand for SARS-CoV-2 testing. The aim of this study is to present a reproducible workflow for real time RT-PCR SARS-CoV-2 testing using OT-2 open-source liquid-handling robots (Opentrons, NY). We have developed a framework that includes a code template which is helpful for building different stand-alone robotic stations, capable of performing specific protocols. Such stations can be combined together to create a complex multi-stage workflow, from sample setup to real time RT-PCR. Using our open-source code, it is easy to create new stations or workflows from scratch, adapt existing templates to update the experimental protocols, or to fine-tune the code to fit specific needs. Using this framework, we developed the code for two different workflows and evaluated them using external quality assessment (EQA) samples from the European Molecular Genetics Quality Network (EMQN). The affordability of this platform makes automated SARS-CoV-2 PCR testing accessible for most laboratories and hospitals with qualified bioinformatics personnel. This platform also allows for flexibility, as it is not dependent on any specific commercial kit, and thus it can be quickly adapted to protocol changes, reagent, consumable shortages, or any other temporary material constraints.

Evaluation of six different rapid methods for nucleic acid detection of SARS-COV-2 virus.

In the current coronavirus disease 2019 (COVID-19) pandemic there is a mass screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) happening around the world due to the extensive spread of the infections. There is a high demand for rapid diagnostic tests to expedite the identification of cases and to facilitate early isolation and control spread. Hence this study evaluates six different rapid nucleic acid detection assays that are commercially available for SARS-CoV-2 virus detection. Nasopharyngeal samples were collected from 4981 participants and were tested for the SARS-CoV-2 virus by the gold standard real-time reverse-transcription polymerase chain reaction (RT-PCR) method and with one of these six rapid methods of detection. Evaluation of the rapid nucleic acid detection assays was done by comparing the results of these rapid methods with the gold standard RT-qPCR results for SARS-COV-2 detection. AQ-TOP had the highest sensitivity (98%) and a strong kappa value of 0.943 followed by Genechecker and Abbot ID NOW. The POCKIT (ii RT-PCR) assay had the highest test accuracy of 99.29% followed by Genechecker and Cobas Liat. Atila iAMP showed the highest percentage of invalid reports (35.5%) followed by AQ-TOP with 6% and POCKIT with 3.7% of invalid reports. Genechecker system, Abbott ID NOW, and Cobas Liat were found to have the best performance and agreement when compared with the standard RT-PCR for COVID-19 detection. With further research, these rapid tests have the potential to be employed in large-scale screening of COVID-19.

RT-PCR/MALDI-TOF mass spectrometry-based detection of SARS-CoV-2 in saliva specimens.

As severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infections continue, there is a substantial need for cost-effective and large-scale testing that utilizes specimens that can be readily collected from both symptomatic and asymptomatic individuals in various community settings. Although multiple diagnostic methods utilize nasopharyngeal specimens, saliva specimens represent an attractive alternative as they can rapidly and safely be collected from different populations. While saliva has been described as an acceptable clinical matrix for the detection of SARS-CoV-2, evaluations of analytic performance across platforms for this specimen type are limited. Here, we used a novel sensitive RT-PCR/MALDI-TOF mass spectrometry-based assay (Agena MassARRAY®) to detect SARS-CoV-2 in saliva specimens. The platform demonstrated high diagnostic sensitivity and specificity when compared to matched patient upper respiratory specimens. We also evaluated the analytical sensitivity of the platform and determined the limit of detection of the assay to be 1562.5 copies/ml. Furthermore, across the five individual target components of this assay, there was a range in analytic sensitivities for each target with the N2 target being the most sensitive. Overall, this system also demonstrated comparable performance when compared to the detection of SARS-CoV-2 RNA in saliva by the cobas® 6800/8800 SARS-CoV-2 real-time RT-PCR Test (Roche). Together, we demonstrate that saliva represents an appropriate matrix for SARS-CoV-2 detection on the novel Agena system as well as on a conventional real-time RT-PCR assay. We conclude that the MassARRAY® system is a sensitive and reliable platform for SARS-CoV-2 detection in saliva, offering scalable throughput in a large variety of clinical laboratory settings.

Comparison of saliva with oral and nasopharyngeal swabs for SARS-CoV-2 detection on various commercial and laboratory-developed assays.

The accurate laboratory detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a crucial element in the fight against coronavirus disease 2019 (COVID-19). Reverse transcription-polymerase chain reaction testing on combined oral and nasopharyngeal swab (ONPS) suffers from several limitations, including the need for qualified personnel, the discomfort caused by invasive nasopharyngeal sample collection, and the possibility of swab and transport media shortage. Testing on saliva would represent an advancement. The aim of this study was to compare the concordance between saliva samples and ONPS for the detection of SARS-CoV-2 on various commercial and laboratory-developed tests (LDT). Individuals were recruited from eight institutions in Quebec, Canada, if they had SARS-CoV-2 RNA detected on a recently collected ONPS, and accepted to provide another ONPS, paired with saliva. Assays available in the different laboratories (Abbott RealTime SARS-CoV-2, Cobas® SARS-CoV-2, Simplexa™ COVID-19 Direct, Allplex™ 2019-nCoV, RIDA®GENE SARS-CoV-2, and an LDT preceded by three different extraction methods) were used to determine the concordance between saliva and ONPS results. Overall, 320 tests were run from a total of 125 saliva and ONPS sample pairs. All assays yielded similar sensitivity when saliva was compared to ONPS, with the exception of one LDT (67% vs. 93%). The mean difference in cycle threshold (∆C t ) was generally (but not significantly) in favor of the ONPS for all nucleic acid amplification tests. The maximum mean ∆​​​​​C t was 2.0, while individual ∆C t varied importantly from -17.5 to 12.4. Saliva seems to be associated with sensitivity similar to ONPS for the detection of SARS-CoV-2 by various assays.

Validation of a direct-to-PCR COVID-19 detection protocol utilizing mechanical homogenization: A model for reducing resources needed for accurate testing.

Efficient and effective viral detection methodologies are a critical piece in the global response to COVID-19, with PCR-based nasopharyngeal and oropharyngeal swab testing serving as the current gold standard. With over 100 million confirmed cases globally, the supply chains supporting these PCR testing efforts are under a tremendous amount of stress, driving the need for innovative and accurate diagnostic solutions. Herein, the utility of a direct-to-PCR method of SARS-CoV-2 detection grounded in mechanical homogenization is examined for reducing resources needed for testing while maintaining a comparable sensitivity to the current gold standard workflow of nasopharyngeal and oropharyngeal swab testing. In a head-to-head comparison of 30 patient samples, this initial clinical validation study of the proposed homogenization-based workflow demonstrated significant agreeability with the current extraction-based method utilized while cutting the total resources needed in half.

Real-time SARS-CoV-2 diagnostic and variants tracking over multiple candidates using nanopore DNA sequencing.

Since December 2019, a novel coronavirus responsible for a severe acute respiratory syndrome (SARS-CoV-2) is accountable for a major pandemic situation. The emergence of the B.1.1.7 strain, as a highly transmissible variant has accelerated the world-wide interest in tracking SARS-CoV-2 variants' occurrence. Similarly, other extremely infectious variants, were described and further others are expected to be discovered due to the long period of time on which the pandemic situation is lasting. All described SARS-CoV-2 variants present several mutations within the gene encoding the Spike protein, involved in host receptor recognition and entry into the cell. Hence, instead of sequencing the whole viral genome for variants' tracking, herein we propose to focus on the SPIKE region to increase the number of candidate samples to screen at once; an essential aspect to accelerate diagnostics, but also variants' emergence/progression surveillance. This proof of concept study accomplishes both at once, population-scale diagnostics and variants' tracking. This strategy relies on (1) the use of the portable MinION DNA sequencer; (2) a DNA barcoding and a SPIKE gene-centered variant's tracking, increasing the number of candidates per assay; and (3) a real-time diagnostics and variant's tracking monitoring thanks to our software RETIVAD. This strategy represents an optimal solution for addressing the current needs on SARS-CoV-2 progression surveillance, notably due to its affordable implementation, allowing its implantation even in remote places over the world.

Development of robust isothermal RNA amplification assay for lab-free testing of RNA viruses.

Simple tests of infectiousness that return results in minutes and directly from samples even with low viral loads could be a potential game-changer in the fight against COVID-19. Here, we describe an improved isothermal nucleic acid amplification assay, termed the RICCA (RNA Isothermal Co-assisted and Coupled Amplification) reaction, that consists of a simple one-pot format of 'sample-in and result-out' with a primary focus on the detection of low copy numbers of RNA virus directly from saliva without the need for laboratory processing. We demonstrate our assay by detecting 16S rRNA directly from E. coli cells with a sensitivity as low as 8 CFU/µL and RNA fragments from a synthetic template of SARS-CoV-2 with a sensitivity as low as 1740 copies/µL. We further demonstrate the applicability of our assay for real-time testing at the point of care by designing a closed format for paper-based lateral flow assay and detecting heat-inactivated SARS-COV-2 virus in human saliva at concentrations ranging from 28,000 to 2.8 copies/µL with a total assay time of 15-30 min.

An ultra-portable, self-contained point-of-care nucleic acid amplification test for diagnosis of active COVID-19 infection.

There is currently a high level of demand for rapid COVID-19 tests, that can detect the onset of the disease at point of care settings. We have developed an ultra-portable, self-contained, point-of-care nucleic acid amplification test for diagnosis of active COVID-19 infection, based on the principle of loop mediated isothermal amplification (LAMP). The LAMP assay is 100% sensitive and specific to detect a minimum of 300 RNA copies/reaction of SARS-CoV-2. All of the required sample transportation, lysing and amplification steps are performed in a standalone disposable cartridge, which is controlled by a battery operated, pocket size (6x9x4cm3) unit. The test is easy to operate and does not require skilled personnel. The total time from sample to answer is approximately 35 min; a colorimetric readout indicates positive or negative results. This portable diagnostic platform has significant potential for rapid and effective testing in community settings. This will accelerate clinical decision making, in terms of effective triage and timely therapeutic and infection control interventions.

Development and implementation of a scalable and versatile test for COVID-19 diagnostics in rural communities.

Rapid and widespread testing of severe acute respiratory coronavirus 2 (SARS-CoV-2) is essential for an effective public health response aimed at containing and mitigating the coronavirus disease 2019 (COVID-19) pandemic. Successful health policy implementation relies on early identification of infected individuals and extensive contact tracing. However, rural communities, where resources for testing are sparse or simply absent, face distinctive challenges to achieving this success. Accordingly, we report the development of an academic, public land grant University laboratory-based detection assay for the identification of SARS-CoV-2 in samples from various clinical specimens that can be readily deployed in areas where access to testing is limited. The test, which is a quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based procedure, was validated on samples provided by the state laboratory and submitted for FDA Emergency Use Authorization. Our test exhibits comparable sensitivity and exceeds specificity and inclusivity values compared to other molecular assays. Additionally, this test can be re-configured to meet supply chain shortages, modified for scale up demands, and is amenable to several clinical specimens. Test development also involved 3D engineering critical supplies and formulating a stable collection media that allowed samples to be transported for hours over a dispersed rural region without the need for a cold-chain. These two elements that were critical when shortages impacted testing and when personnel needed to reach areas that were geographically isolated from the testing center. Overall, using a robust, easy-to-adapt methodology, we show that an academic laboratory can supplement COVID-19 testing needs and help local health departments assess and manage outbreaks. This additional testing capacity is particularly germane for smaller cities and rural regions that would otherwise be unable to meet the testing demand.

Oil immersed lossless total analysis system for integrated RNA extraction and detection of SARS-CoV-2.

The COVID-19 pandemic exposed difficulties in scaling current quantitative PCR (qPCR)-based diagnostic methodologies for large-scale infectious disease testing. Bottlenecks include lengthy multi-step processes for nucleic acid extraction followed by qPCR readouts, which require costly instrumentation and infrastructure, as well as reagent and plastic consumable shortages stemming from supply chain constraints. Here we report an Oil Immersed Lossless Total Analysis System (OIL-TAS), which integrates RNA extraction and detection onto a single device that is simple, rapid, cost effective, and requires minimal supplies and infrastructure to perform. We validated the performance of OIL-TAS using contrived SARS-CoV-2 viral particle samples and clinical nasopharyngeal swab samples. OIL-TAS showed a 93% positive predictive agreement (n = 57) and 100% negative predictive agreement (n = 10) with clinical SARS-CoV-2 qPCR assays in testing clinical samples, highlighting its potential to be a faster, cheaper, and easier-to-deploy alternative for infectious disease testing.

Clinical evaluation of a multiplex real-time RT-PCR assay for detection of SARS-CoV-2 in individual and pooled upper respiratory tract samples.

The aim of this study was to identify and validate a sensitive, high-throughput, and cost-effective SARS-CoV-2 real-time RT-PCR assay to be used as a surveillance and diagnostic tool for SARS-CoV-2 in a university surveillance program. We conducted a side-by-side clinical evaluation of a newly developed SARS-CoV-2 multiplex assay (EZ-SARS-CoV-2 Real-Time RT-PCR) with the commercial TaqPath COVID-19 Combo Kit, which has an Emergency Use Authorization from the FDA. The EZ-SARS-CoV-2 RT-PCR incorporates two assays targeting the SARS-CoV-2 N gene, an internal control targeting the human RNase P gene, and a PCR inhibition control in a single reaction. Nasopharyngeal (NP) and anterior nares (AN) swabs were tested as individuals and pools with both assays and in the ABI 7500 Fast and the QuantStudio 5 detection platforms. The analytical sensitivity of the EZ-SARS-CoV-2 RT-PCR assay was 250 copies/ml or approximately 1.75 genome copy equivalents per reaction. The clinical performance of the EZ-SARS-CoV-2 assay was evaluated using NP and AN samples tested in other laboratories. The diagnostic sensitivity of the assay ranged between 94 and 96% across the detection platforms, and the diagnostic specificity was 94.06%. The positive predictive value was 94%, and the negative predictive value ranged from 94 to 96%. Pooling five NP or AN specimens yielded 93% diagnostic sensitivity. The overall agreement between these SARS-CoV-2 RT-PCR assays was high, supported by a Cohen's kappa value of 0.93. The EZ-SARS-CoV-2 RT-PCR assay performance attributes of high sensitivity and specificity with AN sample matrix and pooled upper respiratory samples support its use in a high-throughput surveillance testing program.