SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity

Vogels, Chantal B.F.; Watkins, Anne E.; Harden, Christina A.; Brackney, Douglas E.; Shafer, Jared; Wang, Jianghui; Caraballo, César; Kalinich, Chaney C.; Ott, Isabel M.; Fauver, Joseph R.; Kudo, Eriko; Lu, Peiwen; Venkataraman, Arvind; Tokuyama, Maria; Moore, Adam J.; Muenker, M. Catherine; Casanovas‐Massana, Arnau; Fournier, John; Bermejo, Santos; Campbell, Melissa; Datta, Rupak; Nelson, Allison; Cruz, Charles S. Dela; Ko, Albert Icksang; Iwasaki, Akiko; Krumholz, Harlan M.; Matheus, JD; Hui, Pei; Liu, Chen; Farhadian, Shelli; Sikka, Robby; Grubaugh, Nathan D.

doi:10.1016/j.medj.2020.12.010

Cited by 236 publications

(325 citation statements)

References 51 publications

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“…On testing this workflow on clinical samples, we found a close agreement with the kitbased RNA extraction method, which is used for detection of SARS-CoV-2. Our results were thus consistent with previous reports on the use of RNA extraction-free detection of SARS- CoV-2 (Alcoba-Florez et al, 2020;Brown et al, 2020;Bruce et al, 2020;Grant et al, 2020;Hasan et al, 2020;Merindol et al, 2020;Srivatsan et al, 2020;Vogels et al, 2020b;Wee et al, 2020).…”

Section: Discussionsupporting

confidence: 93%

“…In addition, workflow of these methods is contingent on additional RNA extraction steps that require trained professionals and sophisticated laboratory set-up and instrumentation to perform the assay. Considering these fundamental challenges, we developed, optimized, and validated the use of a simple workflow to detect SARS-CoV-2 in saliva samples for the following reasons: (1) saliva can be self-collected with ease to minimize direct contact with the healthcare workers, and to reduce handling errors; (2) repeated sampling is feasible without incurring discomfort to the patient, with a reasonably uniform sample distribution; and (3) higher stability of the SARS-CoV-2 in saliva even when stored at room temperature (up to 7 days) (Vogels et al, 2020b). Initially, we validated the analytical sensitivity of clinical saliva samples following RNA-extraction and RT-qPCR assay for detection, and found a close agreement with corresponding swab test results performed by the hospital.…”

Section: Discussionmentioning

confidence: 99%

“…The results show comparable sensitivity to RNA extraction methods (Alcoba-Florez et al, 2020;Brown et al, 2020;Bruce et al, 2020;Grant et al, 2020;Hasan et al, 2020;Merindol et al, 2020;Srivatsan et al, 2020;Wee et al, 2020). Similarly, these methods were also optimized on saliva samples (Lalli et al, 2020;Ranoa et al, 2020;Vogels et al, 2020b), although contradictory reports exist regarding sensitivity based on saliva than swab samples for SARS-CoV-2 (Meyerson et al, 2020;Williams et al, 2020;Wyllie et al, 2020). Nevertheless, all these studies unanimously suggest that nucleic acid extraction-free detection of SARS-CoV-2 is feasible.…”

Section: Introductionmentioning

confidence: 94%

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A Saliva-Based RNA Extraction-Free Workflow Integrated With Cas13a for SARS-CoV-2 Detection

Azmi

Faizan

Kumar

et al. 2021

Front. Cell. Infect. Microbiol.

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A major bottleneck in scaling-up COVID-19 testing is the need for sophisticated instruments and well-trained healthcare professionals, which are already overwhelmed due to the pandemic. Moreover, the high-sensitive SARS-CoV-2 diagnostics are contingent on an RNA extraction step, which, in turn, is restricted by constraints in the supply chain. Here, we present CASSPIT (Cas13 Assisted Saliva-based & Smartphone Integrated Testing), which will allow direct use of saliva samples without the need for an extra RNA extraction step for SARS-CoV-2 detection. CASSPIT utilizes CRISPR-Cas13a based SARS-CoV-2 RNA detection, and lateral-flow assay (LFA) readout of the test results. The sample preparation workflow includes an optimized chemical treatment and heat inactivation method, which, when applied to COVID-19 clinical samples, showed a 97% positive agreement with the RNA extraction method. With CASSPIT, LFA based visual limit of detection (LoD) for a given SARS-CoV-2 RNA spiked into the saliva samples was ~200 copies; image analysis-based quantification further improved the analytical sensitivity to ~100 copies. Upon validation of clinical sensitivity on RNA extraction-free saliva samples (n = 76), a 98% agreement between the lateral-flow readout and RT-qPCR data was found (Ct<35). To enable user-friendly test results with provision for data storage and online consultation, we subsequently integrated lateral-flow strips with a smartphone application. We believe CASSPIT will eliminate our reliance on RT-qPCR by providing comparable sensitivity and will be a step toward establishing nucleic acid-based point-of-care (POC) testing for COVID-19.

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Section: Discussionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 94%

See 1 more Smart Citation

A Saliva-Based RNA Extraction-Free Workflow Integrated With Cas13a for SARS-CoV-2 Detection

Azmi

Faizan

Kumar

et al. 2021

Front. Cell. Infect. Microbiol.

View full text Add to dashboard Cite

show abstract

“…Currently, all FDA reviewed kits that use saliva specimens require RNA purification, and their LODs range from 600 to 180,000 virus copies per mL of saliva [19]. Various methods for detecting SARS-CoV-2 in saliva without RNA extraction report LOD values from 1000 to 14,000 copies/mL [7,9,10]. AG processing is effective over a wide range of viral concentrations in either saliva specimens or samples collected by swab into media.…”

Section: Plos Onementioning

confidence: 99%

“…For this method, the LOD of the final RT-PCR mix, evaluated using saliva spiked with a viral RNA template of 99 bp, was 12 copies of RNA template per reaction (equivalent to 14,000 copies of viral RNA template per ml of saliva). Another publication described a method in which saliva samples were treated with proteinase K and then heated before being used in a direct RT-PCR assay [10]. The reported LOD for detecting SARS-CoV-2, evaluated using SARS-CoV-2 positive saliva, was 6,000 virus copies per ml of saliva.…”

Section: Introductionmentioning

confidence: 99%

Rapid processing of SARS-CoV-2 containing specimens for direct RT-PCR

Chomczyński¹,

Chomczynski²,

Kennedy³

et al. 2021

PLoS ONE

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Widespread diagnostic testing is needed to reduce transmission of COVID-19 and manage the pandemic. Effective mass screening requires robust and sensitive tests that reliably detect the SARS-CoV-2 virus, including asymptomatic and pre-symptomatic infections with a low viral count. Currently, the most accurate tests are based on detection of viral RNA by RT-PCR. We developed a method to process COVID-19 specimens that simplifies and increases the sensitivity of viral RNA detection by direct RT-qPCR, performed without RNA purification. In the method, termed Alkaline-Glycol Processing (AG Processing), a SARS-CoV-2-containing biological specimen, such as saliva or a swab-collected suspension, is processed at pH 12.2 to 12.8 for 5 min at room temperature. An aliquot of the AG-processed specimen is used for detection of SARS-CoV-2 RNA by direct RT-qPCR. AG processing effectively lyses viruses and reduces the effect of inhibitors of RT-PCR that are present in biological specimens. The sensitivity of detecting viral RNA using AG processing is on par with methods that include a viral RNA purification step. One copy of SARS-CoV-2 virus per reaction, equivalent to 300 copies per ml of saliva, is detectable in the AG-processed saliva. The LOD is 300 viral copies per ml of initial saliva specimen. AG processing works with saliva specimens or swab specimens collected into Universal Transport Medium, is compatible with heat treatment of saliva, and was confirmed to work with a range of CDC-approved RT-qPCR products and kits. Detection of SARS-CoV-2 RNA using AG processing with direct RT-qPCR provides a reliable and scalable diagnostic test for COVID-19 that can be integrated into a range of workflows, including automated settings.

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Assessment of a Program for SARS-CoV-2 Screening and Environmental Monitoring in an Urban Public School District

et al. 2021

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Key Points Question Does weekly testing of kindergarten through 12th grade students and staff improve detection of SARS-CoV-2 infection and understanding of the epidemiology of SARS-CoV-2 in urban public school settings? Findings In this quality improvement study, weekly school-based saliva polymerase chain reaction testing at 3 urban public schools was associated with increased case detection among staff and students compared with symptom-based strategies, exceeding county-level case rates. SARS-CoV-2 was detected in school wastewater samples each week as well as air and surface samples from choir classrooms. Meaning This study suggests that routine SARS-CoV-2 testing may identify infected staff and students who are not identified through conventional case detection and may provide insight into disease burdens of undertested communities.

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SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity

Cited by 236 publications

References 51 publications

A Saliva-Based RNA Extraction-Free Workflow Integrated With Cas13a for SARS-CoV-2 Detection

A Saliva-Based RNA Extraction-Free Workflow Integrated With Cas13a for SARS-CoV-2 Detection

Rapid processing of SARS-CoV-2 containing specimens for direct RT-PCR

Assessment of a Program for SARS-CoV-2 Screening and Environmental Monitoring in an Urban Public School District

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