CRISPR-Cas12a-Based Detection of SARS-CoV-2 Harboring the E484K Mutation
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ABSTRACT:
SUBMITTER: Marques M
PROVIDER: S-EPMC8610009 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Fast evolving of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has caused the spreading of COVID-19 disease rapidly around the globe. The mutation, especially in the gene encoding spike protein has helped the virus adapt and evade human immune system, as well as affect the efficacy of the immunizations and treatments. SARS-CoV-2 variant carrying D614G amino acid change at the spike protein is the most dominant strain in the pandemic. Therefore, efficient detection of the SARS-CoV-2 variants including D614G mutation is critical to control the COVID-19 pandemic. Herein, we report a dual synthetic mismatches CRISPR/Cas12a (dsmCRISPR) method to detect the SARS-CoV-2 D614G mutation with high sensitivity and specificity. By targeting SARS-CoV-2 D614G mutation, synthetic mismatch crRNAs were designed from -3 to +3 position around the mutation site. To improve the sensitivity and specificity, a synthetic mismatch primer with a 3'-terminal base complementary to the D614G point mutation and a mismatch next to 3'-terminal base was used to specifically amplify the D614G mutation site with higher annealing temperature. Using synthetic mismatch crRNA-(-1), a higher ratio (13.45) of the fluorescence between G614 and D614 was observed. When combined with mismatch primer to amplify D614G mutation, the fluorescence ratio of G614/D164 template detected was increased by 73.53% to 23.12. This method can detect the SARS-CoV-2 D614G mutation nucleic acid with high sensitivity, which was validated with synthetic SARS-CoV-2 D614G RNA. Therefore, the dsmCRIPSR method has significant potential to serve as a sensitive and specific assay for SARS-CoV-2 D614G detection and could be further extended for the detection of other SARS-CoV-2 variants of interest.
| S-EPMC8339451 | biostudies-literature
Project description:The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1-10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.
| S-EPMC7737895 | biostudies-literature
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.
| S-EPMC8597640 | biostudies-literature
Project description:BackgroundCOVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component.MethodsWe tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19.ResultsThe OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input.ConclusionsThe OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.
| S-EPMC7884969 | biostudies-literature
Project description:BackgroundReal-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis.MethodsA Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios.FindingsThe ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative.InterpretationSENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic.FundingDetailed funding information is available at the end of the manuscript.
| S-EPMC7544594 | biostudies-literature
Project description:The outbreak of the SARS-CoV-2 caused the disease COVID-19 to spread globally. Specific and sensitive detection of SARS-CoV-2 facilitates early intervention and prevents the disease from spreading. Here, we present a solid-state CRISPR-Cas12a-assisted nanopore (SCAN) sensing strategy for the specific detection of SARS-CoV-2. We introduced a nanopore-sized counting method to measure the cleavage ratio of reporters, which is used as a criterion for positive/negative classification. A kinetic cleavage model was developed and validated to predict the reporter size distributions. The model revealed the trade-offs between sensitivity, turnaround time, and false-positive rate of the SARS-CoV-2 SCAN. With preamplification and a 30 min CRISPR Cas12a assay, we achieved excellent specificity against other common human coronaviruses and a limit of detection of 13.5 copies/μL (22.5 aM) of viral RNA at a confidence level of 95%. These results suggested that the SCAN could provide a rapid, sensitive, and specific analysis of SARS-CoV-2.
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Project description:The Delta variant of concern of severe acute respiratory syndrome coronavirus 2 is dominant worldwide. We report a case cluster caused by Delta sublineage B.1.617.2 harboring the mutation E484K in Italy during July 11-July 29, 2021. This mutation appears to affect immune response and vaccine efficacy; monitoring its appearance is urgent.
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Project description:Antigen detection provides particularly valuable information for medical diagnoses; however, the current detection methods are less sensitive and accurate than nucleic acid analysis. The combination of CRISPR/Cas12a and aptamers provides a new detection paradigm, but sensitive sensing and stable amplification in antigen detection remain challenging. Here, we present a PCR-free multiple trigger dsDNA tandem-based signal amplification strategy and a de novo designed dual aptamer synergistic sensing strategy. Integration of these two strategies endowed the CRISPR/Cas12a and aptamer-based method with ultra-sensitive, fast, and stable antigen detection. In a demonstration of this method, the limit of detection was at the single virus level (0.17 fM, approximately two copies/μL) in SARS-CoV-2 antigen nucleocapsid protein analysis of saliva or serum samples. The entire procedure required only 20 min. Given our system's simplicity and modular setup, we believe that it could be adapted reasonably easily for general applications in CRISPR/Cas12a-aptamer-based detection.
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