Dataset Information

Equipment-free detection of SARS-CoV-2 and Variants of Concern using Cas13.

ABSTRACT: The COVID-19 pandemic, and the recent rise and widespread transmission of SARS-CoV-2 Variants of Concern (VOCs), have demonstrated the need for ubiquitous nucleic acid testing outside of centralized clinical laboratories. Here, we develop SHINEv2, a Cas13-based nucleic acid diagnostic that combines quick and ambient temperature sample processing and lyophilized reagents to greatly simplify the test procedure and assay distribution. We benchmarked a SHINEv2 assay for SARS-CoV-2 detection against state-of-the-art antigen-capture tests using 96 patient samples, demonstrating 50-fold greater sensitivity and 100% specificity. We designed SHINEv2 assays for discriminating the Alpha, Beta, Gamma and Delta VOCs, which can be read out visually using lateral flow technology. We further demonstrate that our assays can be performed without any equipment in less than 90 minutes. SHINEv2 represents an important advance towards rapid nucleic acid tests that can be performed in any location.

SUBMITTER: Arizti-Sanz J

PROVIDER: S-EPMC8575147 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Equipment-free detection of SARS-CoV-2 and Variants of Concern using Cas13.

Arizti-Sanz Jon J Bradley A'Doriann A Zhang Yibin B YB Boehm Chloe K CK Freije Catherine A CA Grunberg Michelle E ME Kosoko-Thoroddsen Tinna-Solveig F TF Welch Nicole L NL Pillai Priya P PP Mantena Sreekar S Kim Gaeun G Uwanibe Jessica N JN John Oluwagboadurami G OG Eromon Philomena E PE Kocher Gregory G Gross Robin R Lee Justin S JS Hensley Lisa E LE Happi Christian T CT Johnson Jeremy J Sabeti Pardis C PC Myhrvold Cameron C

medRxiv : the preprint server for health sciences 20211102

The COVID-19 pandemic, and the recent rise and widespread transmission of SARS-CoV-2 Variants of Concern (VOCs), have demonstrated the need for ubiquitous nucleic acid testing outside of centralized clinical laboratories. Here, we develop SHINEv2, a Cas13-based nucleic acid diagnostic that combines quick and ambient temperature sample processing and lyophilized reagents to greatly simplify the test procedure and assay distribution. We benchmarked a SHINEv2 assay for SARS-CoV-2 detection against ...[more]

PMID: 34751276

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Project description:Throughout the coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continuously evolved, resulting in new variants, some of which possess increased infectivity, immune evasion, and virulence. Such variants have been denoted by the World Health Organization as variants of concern (VOC) because they have resulted in an increased number of cases, posing a strong risk to public health. Thus far, five VOCs have been designated, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529), including their sublineages. Next-generation sequencing (NGS) can produce a significant amount of information facilitating the study of variants; however, NGS is time-consuming and costly and not efficient during outbreaks, when rapid identification of VOCs is urgently needed. In such periods, there is a need for fast and accurate methods, such as real-time reverse transcription PCR in combination with probes, which can be used for monitoring and screening of the population for these variants. Thus, we developed a molecular beacon-based real-time RT-PCR assay according to the principles of spectral genotyping. This assay employs five molecular beacons that target ORF1a:ΔS3675/G3676/F3677, S:ΔH69/V70, S:ΔE156/F157, S:ΔΝ211, S:ins214EPE, and S:ΔL242/A243/L244, deletions and an insertion found in SARS-CoV-2 VOCs. This assay targets deletions/insertions because they inherently provide higher discrimination capacity. Here, the design process of the molecular beacon-based real-time RT-PCR assay for detection and discrimination of SARS-CoV-2 is presented, and experimental testing using SARS-CoV-2 VOC samples from reference strains (cultured virus) and clinical patient samples (nasopharyngeal samples), which have been previously classified using NGS, were evaluated. Based on the results, it was shown that all molecular beacons can be used under the same real-time RT-PCR conditions, consequently improving the time and cost efficiency of the assay. Furthermore, this assay was able to confirm the genotype of each of the tested samples from various VOCs, thereby constituting an accurate and reliable method for VOC detection and discrimination. Overall, this assay is a valuable tool that can be used for screening and monitoring the population for VOCs or other emerging variants, contributing to limiting their spread and protecting public health.

Project description:A big challenge for the control of COVID-19 pandemic is the emergence of variants of concern (VOCs) or variants of interest (VOIs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be more transmissible and/or more virulent and could escape immunity obtained through infection or vaccination. A simple and rapid test for SARS-CoV-2 variants is an unmet need and is of great public health importance. In this study, we designed and analytically validated a CRISPR-Cas12a system for direct detection of SARS-CoV-2 VOCs. We further evaluated the combination of ordinary reverse transcription-PCR (RT-PCR) and CRISPR-Cas12a to improve the detection sensitivity and developed a universal system by introducing a protospacer adjacent motif (PAM) near the target mutation sites through PCR primer design to detect mutations without PAM. Our results indicated that the CRISPR-Cas12a assay could readily detect the signature spike protein mutations (K417N/T, L452R/Q, T478K, E484K/Q, N501Y, and D614G) to distinguish alpha, beta, gamma, delta, kappa, lambda, and epsilon variants of SARS-CoV-2. In addition, the open reading frame 8 (ORF8) mutations (T/C substitution at nt28144 and the corresponding change of amino acid L/S) could differentiate L and S lineages of SARS-CoV-2. The low limit of detection could reach 10 copies/reaction. Our assay successfully distinguished 4 SARS-CoV-2 strains of wild type and alpha (B.1.1.7), beta (B.1.351), and delta (B.1.617.2) variants. By testing 32 SARS-CoV-2-positive clinical samples infected with the wild type (n = 5) and alpha (n = 11), beta (n = 8), and delta variants (n = 8), the concordance between our assay and sequencing was 100%. The CRISPR-based approach is rapid and robust and can be adapted for screening the emerging mutations and immediately implemented in laboratories already performing nucleic acid amplification tests or in resource-limited settings. IMPORTANCE We described CRISPR-Cas12-based multiplex allele-specific assay for rapid SARS-CoV-2 variant genotyping. The new system has the potential to be quickly developed, continuously updated, and easily implemented for screening of SARS-CoV-2 variants in resource-limited settings. This approach can be adapted for emerging mutations and implemented in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests using existing resources and extracted nucleic acid.

Dataset Information

Equipment-free detection of SARS-CoV-2 and Variants of Concern using Cas13.

Publications

Equipment-free detection of SARS-CoV-2 and Variants of Concern using Cas13.

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