Detection of SARS-CoV-2 with Solid-State CRISPR-Cas12a-Assisted Nanopores.
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ABSTRACT:
SUBMITTER: Nouri R
PROVIDER: S-EPMC8491552 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Solid-state nanopores have emerged as promising platforms for biosensing including diagnostics for disease detection. Here we show nanopore experiments that detect CRISPR-dCas9, a sequence-specific RNA-guided protein system that specifically binds to a target DNA sequence. While CRISPR-Cas9 is acclaimed for its gene editing potential, the CRISPR-dCas9 variant employed here does not cut DNA but instead remains tightly bound at a user-defined binding site, thus providing an excellent target for biosensing. In our nanopore experiments, we observe the CRISPR-dCas9 proteins as local spikes that appear on top of the ionic current blockade signal of DNA molecules that translocate through the nanopore. The proteins exhibit a pronounced blockade signal that allows for facile identification of the targeted sequence. Even at the high salt conditions (1 M LiCl) required for nanopore experiments, dCas9 proteins are found to remain stably bound. The binding position of the target sequence can be read from the spike position along the DNA signal. We anticipate applications of this nanopore-based CRISPR-dCas9 biosensing approach in DNA-typing based diagnostics such as quick disease-strain identification, antibiotic-resistance detection, and genome typing.
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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.
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Project description:The current pandemic of COVID-19 caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) has raised significant public health concerns. Rapid and accurate testing of SARS-CoV-2 is urgently needed for early detection and control of the disease spread. Here, we present an RT-LAMP coupled glass nanopore digital counting method for rapid detection of SARS-CoV-2. We validated and compared two one-pot RT-LAMP assays targeting nucleocapsid (N) and envelop (E) genes. The nucleocapsid assay was adopted due to its quick time to positive and better copy number sensitivity. For qualitative positive/negative classification of a testing sample, we used the glass nanopore to digitally count the RT-LAMP amplicons and benchmarked the event rate with a threshold. Due to its intrinsic single molecule sensitivity, nanopore sensors could capture the amplification dynamics more rapidly (quick time to positive). We validated our RT-LAMP coupled glass nanopore digital counting method for SARS-CoV-2 detection by using both spiked saliva samples and COVID-19 clinical nasopharyngeal swab samples. The results obtained showed excellent agreement with the gold standard RT-PCR assay. With its integration capability, the electronic nanopore digital counting platform has significant potential to provide a rapid, sensitive, and specific point-of-care assay for SARS-CoV-2.
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Project description:The novel respiratory virus SARS-CoV-2 is rapidly evolving across the world with the
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Project description:Using nanopores for single-molecule sequencing of proteins - similar to nanopore-based sequencing of DNA - faces multiple challenges, including unfolding of the complex tertiary structure of the proteins and enforcing their unidirectional translocation through nanopores. Here, we combine molecular dynamics (MD) simulations with single-molecule experiments to investigate the utility of SDS (Sodium Dodecyl Sulfate) to unfold proteins for solid-state nanopore translocation, while simultaneously endowing them with a stronger electrical charge. Our simulations and experiments prove that SDS-treated proteins show a considerable loss of the protein structure during the nanopore translocation. Moreover, SDS-treated proteins translocate through the nanopore in the direction prescribed by the electrophoretic force due to the negative charge impaired by SDS. In summary, our results suggest that SDS causes protein unfolding while facilitating protein translocation in the direction of the electrophoretic force; both characteristics being advantageous for future protein sequencing applications using solid-state nanopores.
| S-EPMC5611827 | 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:[This corrects the article DOI: 10.1038/s41421-018-0028-z.].
| S-EPMC6411867 | 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: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.
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Project description:We report an experimental study of using DNA translocation through solid-state nanopores to detect the sequential arrangement of two double-stranded 12-mer hybridization segments on a single-stranded DNA molecule. The sample DNA is a trimer molecule formed by hybridizing three single-stranded oligonucleotides. A polystyrene bead is attached to the end of the trimer DNA, providing a mechanism in slowing down the translocation and suppressing the thermal diffusion, thereby allowing the detection of short features of DNA by standard patch-clamp electronics. The electrical signature of the translocation of a trimer molecule through a nanopore has been identified successfully in the temporal traces of ionic current. The results reported here represent the first successful attempt in using a solid-state nanopore as an ionic scanning device in resolving individual hybridization segments (or 'probes') on a DNA molecule.
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