Harnessing recombinase polymerase amplification for rapid multi-gene detection of SARS-CoV-2 in resource-limited settings

Cherkaoui, Dounia; Da, Huang; Miller, Benjamin S.; Turbé, Valérian; McKendry, Rachel A.

doi:10.1016/j.bios.2021.113328

Cited by 46 publications

(32 citation statements)

References 37 publications

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“…At present, nucleic acid-based test is most popular towards the diagnosis of COVID-19 ( Cherkaoui et al, 2021 ; Yüce et al, 2021 ). Here the detection principle majorly encounters the complex requirement of amplification of cDNA extracted from the viral SARS-CoV-2 RNA collected from bio-fluids of patients under observation ( W. H. Feng et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

A rapid RNA extraction-free lateral flow assay for molecular point-of-care detection of SARS-CoV-2 augmented by chemical probes

Dighe

Moitra

Alafeef

et al. 2022

Biosensors and Bioelectronics

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The coronavirus disease 2019 (COVID-19) pandemic has highlighted the major shortcoming of healthcare systems globally in their inability to diagnose the disease rapidly and accurately. At present, the molecular approaches for diagnosing COVID-19 primarily use reverse transcriptase to create and amplify cDNA from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. Although molecular tests are reported to be specific, false negatives are quite common. Furthermore, literally all these tests require a step involving RNA isolation which does not make them point-of-care (POC) in the true sense. Here, we report a lateral flow strip-based RNA extraction and amplification-free nucleic acid test (NAT) for rapid diagnosis of positive COVID-19 cases at POC. The assay uses highly specific 6-carboxyfluorescein (6-FAM) and biotin labeled antisense oligonucleotides (ASOs) as probes those are designed to target N-gene sequence of SARS-CoV-2. Additionally, we utilized cysteamine capped gold-nanoparticles (Cyst-AuNPs) to augment the signal further for enhanced sensitivity. Without any large-stationary equipment and highly trained staffers, the entire sample-to-answer approach in our case would take less than 30 min from a patient swab sample collection to final diagnostic result. Moreover, when evaluated with 60 clinical samples and verified with an FDA-approved TaqPath RT-PCR kit for COVID-19 diagnosis, the assay obtained almost 99.99% accuracy and specificity. We anticipate that the newly established low-cost amplification-free detection of SARS-CoV-2 RNA will aid in the development of a platform technology for rapid and POC diagnosis of COVID-19 and other pathogens.

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Section: Introductionmentioning

confidence: 99%

A rapid RNA extraction-free lateral flow assay for molecular point-of-care detection of SARS-CoV-2 augmented by chemical probes

Dighe

Moitra

Alafeef

et al. 2022

Biosensors and Bioelectronics

View full text Add to dashboard Cite

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“…Differential target amplification in isothermal reactions typically stems from unpredictable secondary structures in the target RNA, secondary structure in the primer ssDNA, primer dimerization, primer hybridization to each other, and improper hybridization of the primer to the RNA at low temperature (RT-RPA working temperature 37–42 °C) and has been previously observed for RPA-mediated amplification of SARS-CoV-2. 50 , 51 …”

Section: Resultsmentioning

confidence: 99%

Bio-SCAN: A CRISPR/dCas9-Based Lateral Flow Assay for Rapid, Specific, and Sensitive Detection of SARS-CoV-2

et al. 2021

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Simple, rapid, specific, and sensitive point-of-care detection methods are needed to contain the spread of SARS-CoV-2. CRISPR/Cas9-based lateral flow assays are emerging as a powerful alternative for COVID-19 diagnostics. Here, we developed Bio-SCAN (biotin-coupled specific CRISPR-based assay for nucleic acid detection) as an accurate pathogen detection platform that requires no sophisticated equipment or technical expertise. Bio-SCAN detects the SARS-CoV-2 genome in less than 1 h from sample collection to result. In the first step, the target nucleic acid sequence is isothermally amplified in 15 min via recombinase polymerase amplification before being precisely detected by biotin-labeled nuclease-dead SpCas9 (dCas9) on commercially available lateral flow strips. The resulting readout is visible to the naked eye. Compared to other CRISPR-Cas-based pathogen detection assays, Bio-SCAN requires no additional reporters, probes, enhancers, reagents, or sophisticated devices to interpret the results. Bio-SCAN is highly sensitive and successfully detected a clinically relevant level (4 copies/μL) of synthetic SARS-CoV-2 RNA genome. Similarly, Bio-SCAN showed 100% negative and 96% positive predictive agreement with RT-qPCR results when using clinical samples (86 nasopharyngeal swab samples). Furthermore, incorporating variant-specific sgRNAs in the detection reaction allowed Bio-SCAN to efficiently distinguish between the α, β, and δ SARS-CoV-2 variants. Also, our results confirmed that the Bio-SCAN reagents have a long shelf life and can be assembled locally in nonlaboratory and limited-resource settings. Furthermore, the Bio-SCAN platform is compatible with the nucleic acid quick extraction protocol. Our results highlight the potential of Bio-SCAN as a promising point-of-care diagnostic platform that can facilitate low-cost mass screening for SARS-CoV-2.

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“…For example, loop-mediated isothermal amplification (RT-LAMP) [34] and recombinase polymerase amplification (RPA) [35] assays have been deployed in laboratories and remote settings, and the use of CRISPR/CAS for pointof-care applications has also been demonstrated [36]. Technologies demonstrated for COVID-19 screening include RT-LAMP [37], RPA [38], and CRISPR/CAS [39,40] systems. The power of CRISPR-based diagnostics is the potential for developing a priori assays quicker and eventual programmable/on-demand assays.…”

Section: Discussionmentioning

confidence: 99%

End-User Perspectives on Using Quantitative Real-Time PCR and Genomic Sequencing in the Field

et al. 2022

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Quantitative real-time PCR and genomic sequencing have become mainstays for performing molecular detection of biological threat agents in the field. There are notional assessments of the benefits, disadvantages, and challenges that each of these technologies offers according to findings in the literature. However, direct comparison between these two technologies in the context of field-forward operations is lacking. Most market surveys, whether published in print form or provided online, are directed to product manufacturers who can address their respective specifications and operations. One method for comparing these technologies is surveying end-users who are best suited for discussing operational capabilities, as they have hands-on experience with state-of-the-art molecular detection platforms and protocols. These end-users include operators in military defense and first response, as well as various research scientists in the public sector such as government and service laboratories, private sector, and civil society such as academia and nonprofit organizations performing method development and executing these protocols in the field. Our objective was to initiate a survey specific to end-users and their feedback. We developed a questionnaire that asked respondents to (1) determine what technologies they currently use, (2) identify the settings where the technologies are used, whether lab-based or field-forward, and (3) rate the technologies according to a set list of criteria. Of particular interest are assessments of sensitivity, specificity, reproducibility, scalability, portability, and discovery power. This article summarizes the findings from the end-user perspective, highlighting technical and operational challenges.

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Harnessing recombinase polymerase amplification for rapid multi-gene detection of SARS-CoV-2 in resource-limited settings

Cited by 46 publications

References 37 publications

A rapid RNA extraction-free lateral flow assay for molecular point-of-care detection of SARS-CoV-2 augmented by chemical probes

A rapid RNA extraction-free lateral flow assay for molecular point-of-care detection of SARS-CoV-2 augmented by chemical probes

Bio-SCAN: A CRISPR/dCas9-Based Lateral Flow Assay for Rapid, Specific, and Sensitive Detection of SARS-CoV-2

End-User Perspectives on Using Quantitative Real-Time PCR and Genomic Sequencing in the Field

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