Overcoming challenges in human saliva gene expression measurements

Ostheim, Patrick; Tichý, Aleš; Sirák, Igor; Davídková, Marie; Stastna, Marketa; Kultova, Gabriela; Paunesku, Tatjana; Woloschak, Gayle E.; Majewski, Matthäus; Port, Matthias; Abend, Michael

doi:10.1038/s41598-020-67825-6

Cited by 35 publications

(38 citation statements)

References 26 publications

(32 reference statements)

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“…The LoD which we obtained for extraction free saliva samples with a two-pot reaction is slightly lower than what others found when RNA-extraction methods were used (Patchsung et al, 2020). This could be due to: (1) High DNA, proteins, and other ions present in saliva, which interferes with the detection, irrespective of the type of detection method used, i.e., RT-qPCR or SHERLOCK (Ochert et al, 1994;Ostheim et al, 2020). (2) Ambiguity in visual detection, when the test lane signal is shallow and difficult to differentiate by the naked eye.…”

Section: Discussioncontrasting

confidence: 62%

“…Next, we developed a simple workflow to detect SARS-CoV-2 in saliva samples without need for an extra and time-consuming RNAextraction step. Optimization of this workflow was challenging as unlike other biological fluids, the molecular composition of saliva hinders RNA detection, besides being more amenable to RNases (Ochert et al, 1994;Ostheim et al, 2020). Also, SARS-CoV-2 being an enveloped virus, the RNA release condition had to be optimized so that its degradation is minimized.…”

Section: Discussionmentioning

confidence: 99%

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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: Discussioncontrasting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

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

“…With an amplification and detection protocol in place, we next optimized sample processing to establish a simple protocol of heat paired with chemical reagents to promote viral inactivation and dampen the activity of RNA-degrading nucleases present in saliva (Ostheim et al 2020). We assayed two concentrations of the shelf-stable reducing agent TCEP (Tris(2-carboxyethyl)phosphine) paired with the ion chelator EDTA (ethylenediaminetetraacetic acid) (Rabe and Cepko 2020; Myhrvold et al 2018), commercially available reagents such as QuickExtract buffers containing detergents and proteinase, and DNA/RNA shield containing chaotropic guanidine thiocyanate.…”

Section: Resultsmentioning

confidence: 99%

Rapid, point-of-care molecular diagnostics with Cas13

Agrawal

Fanton

Chandrasekaran

et al. 2020

Preprint

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To combat disease outbreaks such as the COVID-19 pandemic, flexible diagnostics for rapid viral detection are greatly needed. We report a nucleic acid test that integrates distinct mechanisms of DNA and RNA amplification optimized for high sensitivity and rapid kinetics, linked to Cas13 detection for specificity. We paired this workflow, termed Diagnostics with Coronavirus Enzymatic Reporting (DISCoVER), with extraction-free sample lysis using shelf-stable reagents that are widely available at low cost. DISCoVER has been validated on saliva samples to incentivize frequent testing for more widespread community surveillance and robustly detected attomolar levels of SARS-CoV-2 within 30 minutes, while avoiding false positives in virus-negative saliva. Furthermore, DISCoVER is compatible with multiplexed CRISPR probes to enable simultaneous detection of a human gene control or alternative pathogens.

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“…This could be due to: (1) Ample amount of DNA, proteins, and other ions present in saliva, which interferes with the detection, irrespective of the type of detection method used, i.e., RT-qPCR or SHERLOCK (Ochert et al, 1994;Ostheim et al, 2020). 2 Thus, to further improve the detection sensitivity of these test results, we employed image-based signal quantification of lateral-flow strips.…”

Section: Discussionmentioning

confidence: 99%

“…The LoD which we obtained for extraction free saliva samples with a two-pot reaction is slightly lower than what others found when RNA-extraction methods were used (Patchsung et al, 2020). This could be due to: (1) Ample amount of DNA, proteins, and other ions present in saliva, which interferes with the detection, irrespective of the type of detection method used, i.e., RT-qPCR or SHERLOCK (Ochert et al, 1994; Ostheim et al, 2020). (2) Ambiguity in visual detection, when the test lane signal is shallow and difficult to differentiate by the naked eye.…”

Section: Discussionmentioning

confidence: 99%

A saliva-based RNA extraction-free workflow integrated with Cas13a for SARS-CoV-2 detection

Azmi¹,

Faizan²,

Kumar

et al. 2020

Preprint

View full text Add to dashboard Cite

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 RNA extraction 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 94 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. 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.

show abstract

Overcoming challenges in human saliva gene expression measurements

Cited by 35 publications

References 26 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, point-of-care molecular diagnostics with Cas13

A saliva-based RNA extraction-free workflow integrated with Cas13a for SARS-CoV-2 detection

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