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Open accessJournal ArticleDOI: 10.3390/V13030420

Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2.

05 Mar 2021-Viruses (Multidisciplinary Digital Publishing Institute)-Vol. 13, Iss: 3, pp 420
Abstract: We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55-100) positive samples and 104/105 (99.05%; 95% CI: 94.81-99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18-99.88) positive samples and 9/9 (100%; 95% CI: 66.37-100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45-36.90) and dilutions of heat-inactivated virus (range: 2.5-100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.

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5 results found


Open accessPosted ContentDOI: 10.1101/2021.04.26.21256081
27 Apr 2021-medRxiv
Abstract: Early detection of SARS-CoV-2 using molecular techniques is paramount to the fight against COVID-19. Due to its high sensitivity and specificity, RT-qPCR is the “gold standard” method for this purpose. However, its technical requirements, processing time and elevated costs hamper its use towards massive and timely molecular testing for COVID-19 in rural and socioeconomically deprived areas of Latin America. The advent and rapid evolution of CRISPR-Cas technology has boosted the development of new pathogen detection methodologies. Recently, DETECTR -a combination of isothermal RT-LAMP amplification and Cas12a-mediated enzymatic detection-has been successfully validated in the Netherlands and the USA as a rapid and low-cost alternative to RT-qPCR for the detection of SARS-CoV-2 from nasopharyngeal swabs. Here, we evaluated the performance of RCSMS, a locally adapted variant of DETECTR, to ascertain the presence of SARS-CoV-2 in saliva samples from 276 patients in two hospitals in Lima, Peru (current status over a total of 350 samples). We show that a low-cost thermochemical treatment with TCEP/EDTA is sufficient to inactivate viral particles and cellular nucleases in saliva, eliminating the need to extract viral RNA with commercial kits, as well as the cumbersome nasopharyngeal swab procedure and the requirement of biosafety level 2 laboratories for molecular analyses. Our clinical validation shows that RCSMS detects up to 5 viral copies per reaction in 40 min, with sensitivity and specificity of 93.8% and 99.0% in the field, respectively, relative to RT-qPCR. Since CRISPR-Cas biosensors can be easily reprogrammed by using different guide RNA molecules, RCSMS has the potential to be quickly adapted for the detection of new SARS-CoV-2 variants. Notably, estimation of its negative and positive predictive values suggests that RCSMS can be confidently deployed in both high and low prevalence settings. Furthermore, our field study validates the use of lateral flow strips to easily visualize the presence of SARS-CoV-2, which paves the way to deploy RCSMS as a “point of care” test in environments with limited access to state-of-the-art diagnostic laboratories. In sum, RCSMS is a fast, efficient and inexpensive alternative to RT-qPCR for expanding COVID-19 testing capacity in low- and middle-income countries.

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1 Citations


Open accessJournal ArticleDOI: 10.1016/J.ISCI.2021.102996
Eric Nalefski, Nidhi Patel, Philip J.Y. Leung, Zeba Islam  +8 moreInstitutions (3)
18 Aug 2021-iScience
Abstract: Bacterial CRISPR systems provide acquired immunity against invading nucleic acids by activating RNA-programmable RNases and DNases. Cas13a and Cas12a enzymes bound to CRISPR RNA (crRNA) recognize specific nucleic acid targets, initiating cleavage of the targets as well as non-target (trans) nucleic acids. Here, we examine the kinetics of single-turnover target and multi-turnover trans-nuclease activities of both enzymes. High-turnover, non-specific Cas13a trans-RNase activity is coupled to rapid binding of target RNA. By contrast, low-turnover Cas12a trans-nuclease activity is coupled to relatively slow cleavage of target DNA, selective for DNA over RNA, indifferent to base identity, and preferential for single-stranded substrates. Combining multiple crRNA increases detection sensitivity of targets, an approach we use to quantify pathogen DNA in samples from patients suspected of Buruli ulcer disease. Results reveal that these enzymes are kinetically adapted to play distinct roles in bacterial adaptive immunity and show how kinetic analysis can be applied to CRISPR-based diagnostics.

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Topics: Trans-activating crRNA (72%), CRISPR (62%), Nucleic acid (56.99%) ... show more

1 Citations


Open accessJournal ArticleDOI: 10.3390/DIAGNOSTICS11071270
15 Jul 2021-
Abstract: The emergence and rapid proliferation of Coronavirus Disease-2019, throughout the past year, has put an unprecedented strain on the global schema of health infrastructure and health economy. The time-sensitive agenda of identifying the virus in humans and delivering a vaccine to the public constituted an effort to flatten the statistical curve of viral spread as it grew exponentially. At the forefront of this effort was an exigency of developing rapid and accurate diagnostic strategies. These have emerged in various forms over the past year-each with strengths and weaknesses. To date, they fall into three categories: (1) those isolating and replicating viral RNA in patient samples from the respiratory tract (Nucleic Acid Amplification Tests; NAATs), (2) those detecting the presence of viral proteins (Rapid Antigen Tests; RATs) and serology-based exams identifying antibodies to the virus in whole blood and serum. The latter vary in their detection of immunoglobulins of known prevalence in early-stage and late-stage infection. With this review, we delineate the categories of testing measures developed to date, analyze the efficacy of collecting patient specimens from diverse regions of the respiratory tract, and present the up and coming technologies which have made pathogen identification easier and more accessible to the public.

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Topics: Coronavirus (51%)

1 Citations


Posted ContentDOI: 10.1101/2021.10.29.466394
29 Oct 2021-bioRxiv
Abstract: Implementing effective monitoring strategies is fundamental to protect crops from pathogens and to ensure the food supply as the world population continues to grow. This is especially important for emergent plant pathogens such as tomato brown rugose fruit virus (ToBRFV), which overcomes the genetic resistance resources used in tomato breeding against tobamoviruses and has become pandemic in less than a decade. Here we report the development of a CRISPR/Cas12a-based test to detect ToBRFV in the laboratory and potentially in a field setting. Using different tobamoviruses to assess specificity, our test showed a clear positive signal for ToBRFV-infected samples, while no cross-reactivity was observed for closely related viruses. Next, we compared the limit of detection of our CRISPR-based test with a reference real-time quantitative PCR test widely used, revealing similar sensitivities for both tests. Finally, to reduce complexity and achieve field-applicability, we used a fast nucleic acid purification step and compared its results side by side with those of a commonly used column-mediated protocol. The new protocol saved time and resources but at the expense of sensitivity. However, it still may be useful to confirm ToBRFV, detection in samples with incipient symptoms of infection. Although there is room for improvement, to our knowledge this is the first field-compatible CRISPR-based test to detect ToBRFV, which combines isothermal amplification with a simplified nucleic acid extraction protocol.

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Open accessJournal ArticleDOI: 10.1128/SPECTRUM.01017-21
Yuanhao Liang, Hongqing Lin, Lirong Zou, Jianhui Zhao  +7 moreInstitutions (1)
Abstract: 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.

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References
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13 results found


Open accessJournal ArticleDOI: 10.1038/S41564-020-0695-Z
Abstract: The present outbreak of a coronavirus-associated acute respiratory disease called coronavirus disease 19 (COVID-19) is the third documented spillover of an animal coronavirus to humans in only two decades that has resulted in a major epidemic. The Coronaviridae Study Group (CSG) of the International Committee on Taxonomy of Viruses, which is responsible for developing the classification of viruses and taxon nomenclature of the family Coronaviridae, has assessed the placement of the human pathogen, tentatively named 2019-nCoV, within the Coronaviridae. Based on phylogeny, taxonomy and established practice, the CSG recognizes this virus as forming a sister clade to the prototype human and bat severe acute respiratory syndrome coronaviruses (SARS-CoVs) of the species Severe acute respiratory syndrome-related coronavirus, and designates it as SARS-CoV-2. In order to facilitate communication, the CSG proposes to use the following naming convention for individual isolates: SARS-CoV-2/host/location/isolate/date. While the full spectrum of clinical manifestations associated with SARS-CoV-2 infections in humans remains to be determined, the independent zoonotic transmission of SARS-CoV and SARS-CoV-2 highlights the need for studying viruses at the species level to complement research focused on individual pathogenic viruses of immediate significance. This will improve our understanding of virus–host interactions in an ever-changing environment and enhance our preparedness for future outbreaks.

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Topics: Coronavirus (64%), Nidovirales (50%)

3,790 Citations


Open accessJournal ArticleDOI: 10.1126/SCIENCE.AAM9321
28 Apr 2017-Science
Abstract: Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a “collateral effect” of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications.

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1,065 Citations


Open accessJournal ArticleDOI: 10.1038/S41587-020-0513-4
Abstract: An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with SARS-CoV-2 infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR–Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA extracts. We validated our method using contrived reference samples and clinical samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT–PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement. SARS-CoV-2 in patient samples is detected in under an hour using a CRISPR-based lateral flow assay.

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860 Citations


Open accessJournal ArticleDOI: 10.1126/SCIENCE.AAS8836
27 Apr 2018-Science
Abstract: Mitigating global infectious disease requires diagnostic tools that are sensitive, specific, and rapidly field deployable. In this study, we demonstrate that the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations as low as 1 copy per microliter. We developed HUDSON (heating unextracted diagnostic samples to obliterate nucleases), a protocol that pairs with SHERLOCK for viral detection directly from bodily fluids, enabling instrument-free DENV detection directly from patient samples in

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Topics: Dengue virus (51%)

531 Citations


Open accessJournal ArticleDOI: 10.1016/J.JMOLDX.2020.03.006
Abstract: The coronavirus disease 2019 (COVID-19) pandemic now has >2,000,000 confirmed cases worldwide. COVID-19 is currently diagnosed using quantitative RT-PCR methods, but the capacity of quantitative RT-PCR methods is limited by their requirement of high-level facilities and instruments. We developed and evaluated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays to detect genomic RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative virus of COVID-19. RT-LAMP assays reported in this study can detect as low as 100 copies of SARS-CoV-2 RNA. Cross-reactivity of RT-LAMP assays to other human coronaviruses was not observed. A colorimetric detection method was adapted for this RT-LAMP assay to enable higher throughput.

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228 Citations