A smartphone-read ultrasensitive and quantitative saliva test for COVID-19

Ning, Bo; Yu, Tao; Zhang, Shengwei; Huang, Zhen; Tian, Di; Lin, Zhen; Niu, Alex; Golden, Nadia; Hensley, Krystle; Threeton, Breanna; Lyon, Christopher J.; Yin, Xiao‐Ming; Roy, Chad J.; Saba, Nakhle S.; Rappaport, Jay; Wei, Qingshan; Hu, Tony

doi:10.1126/sciadv.abe3703

Cited by 202 publications

(171 citation statements)

References 39 publications

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“…Further, owing to the high-sensitivity and highspecificity of Cas13a, our optimized workflow on saliva samples could provide a rapid and better alternative to the existing detection methods and speed up the testing. We envision that in coming time, CRISPR Diagnostics based on either Cas13a or a similar method which utilizes Cas12 (Broughton et al, 2020;Nguyen et al, 2020) and integrated with a smartphone-based readout (Ning et al, 2020) will be a better alternative to RT-qPCR based testing under resource limiting settings. Moreover, integrating CRISPR Diagnostics with RNA extraction-free workflow and smartphone application will provide an alternative to error-prone rapid antigen tests to scale-up COVID-19 testing across resourceconstrained areas and intensify trace, test, and treat strategy for COVID-19.…”

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.

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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.

show abstract

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

“…Further, owing to the high-sensitivity and high-specificity of Cas13a, our optimized workflow on saliva samples could provide a rapid and better alternative to the existing detection methods and speed up the testing. We envision that in coming time, CRISPR-Diagnostics based on either Cas13a or a similar method which utilizes Cas12 (Broughton et al, 2020; Nguyen et al, 2020) and integrated with a smartphone-based readout (Ning et al, 2020) will be a better alternative to RT-qPCR based testing under resource limiting settings. Moreover, integrating CRISPR-Diagnostics with RNA extraction-free workflow and smartphone application will provide an alternative to error-prone rapid antigen tests to scale-up COVID-19 testing across resource-constrained areas and intensify trace, test, and treat strategy for COVID-19.…”

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

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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 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.

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“…Another advantage of using CRISPR/Cas systems is the exclusion of RNA isolation and amplification, making it a faster analysis method. An ultrasensitive RT-RPA CRISPR–fluorescence detection system (FDS) assay can eliminate the need for RNA isolation for SARS-CoV-2 detection [ 54 ]. It uses a saliva sample that is subject to a mix of chemicals that amplify the viral RNA, which is then subjected to CRISPR/Cas12a-based fluorescence signal amplification.…”

Section: Crispr/cas-based Sensors: the New Alternativementioning

confidence: 99%

Point-of-Care PCR Assays for COVID-19 Detection

et al. 2021

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Molecular diagnostics has been the front runner in the world’s response to the COVID-19 pandemic. Particularly, reverse transcriptase-polymerase chain reaction (RT-PCR) and the quantitative variant (qRT-PCR) have been the gold standard for COVID-19 diagnosis. However, faster antigen tests and other point-of-care (POC) devices have also played a significant role in containing the spread of SARS-CoV-2 by facilitating mass screening and delivering results in less time. Thus, despite the higher sensitivity and specificity of the RT-PCR assays, the impact of POC tests cannot be ignored. As a consequence, there has been an increased interest in the development of miniaturized, high-throughput, and automated PCR systems, many of which can be used at point-of-care. This review summarizes the recent advances in the development of miniaturized PCR systems with an emphasis on COVID-19 detection. The distinct features of digital PCR and electrochemical PCR are detailed along with the challenges. The potential of CRISPR/Cas technology for POC diagnostics is also highlighted. Commercial RT–PCR POC systems approved by various agencies for COVID-19 detection are discussed.

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A smartphone-read ultrasensitive and quantitative saliva test for COVID-19

Cited by 202 publications

References 39 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

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

Point-of-Care PCR Assays for COVID-19 Detection

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