EasyCatch, a convenient, sensitive and specific CRISPR detection system for cancer gene mutations.
Ontology highlight
ABSTRACT:
SUBMITTER: Liu Y
PROVIDER: S-EPMC8638196 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:ObjectivesCRISPR-Cas13a system-based nucleic acid detection methods are reported to have rapid and sensitive DNA detection. However, the screening strategy for crRNAs that enables CRISPR-Cas13a single-base resolution DNA detection of human pathogens remains unclear.MethodsA combined rational design and target mutation-anchoring CRISPR RNA (crRNA) screening strategy was proposed.ResultsA set of crRNAs was found to enable the CRISPR-Cas13 system to dramatically distinguish fluroquinolone resistance mutations in clinically isolated Mycobacterium tuberculosis strains from the highly homologous wild type, with a signal ratio ranging from 8.29 to 38.22 in different mutation sites. For the evaluation of clinical performance using genomic DNA from clinically isolated M. tuberculosis, the specificity and sensitivity were 100 and 91.4%, respectively, compared with culture-based phenotypic assays.ConclusionThese results demonstrated that the CRISPR-Cas13a system has potential for use in single nucleotide polymorphism (SNP) detection after tuning crRNAs. We believe this crRNA screening strategy will be used extensively for early drug resistance monitoring and guidance for clinical treatment.
| S-EPMC9136396 | biostudies-literature
Project description:Mobilized colistin resistance (mcr-1) gene mediated by plasmid can cause the speediness dissemination of colistin-resistant strains, which have given rise to a great threat to the treatment of human infection. Hence, a rapid and accurate diagnosis technology for detecting mcr-1 is essential for the control of resistance gene. Here, a recombinase polymerase amplification (RPA) coupled with CRISPR/Cas12a platform was established for rapid, sensitive, and specific detection of mcr-1 gene. The analytical sensitivity of our assay is 420 fg per reaction in pure mcr-1-positive isolates, and the threshold of this method in spiked clinical samples was down to 1.6 × 103 ~ 6.2 × 103 CFU/mL (1.6 ~ 6.2 CFU/reaction). Moreover, the RPA-CRISPR/Cas12a system perspicuously demonstrated no cross-reactivity with other resistant genes. The entire experimental process included rapid DNA extraction (15 min), RPA reaction (30 min), CRISPR/Cas12a cleavage (5 min), and fluorescence testing (<10 min), which could be completed within 60 min. In summary, the RPA-CRISPR/Cas12a assay designed here provides a rapid diagnostic way for monitoring mcr-1 in clinic and livestock farm. IMPORTANCE This study promises a rapid and accurate assay (RPA-CRISPR/Cas12a) for the surveillance of mcr-1 gene, which causes the efficacy loss of colistin in clinical treatments. In addition, the established method is fit for "on-site" surveillance especially.
| S-EPMC9602551 | biostudies-literature
Project description:Hepatitis B remains a major global public health challenge, with particularly high prevalence in medically disadvantaged western Pacific and African regions. Although clinically available technologies for the qPCR detection of HBV are well established, research on point-of-care testing has not progressed substantially. The development of a rapid, accurate point-of-care test is essential for the prevention and control of hepatitis B in medically disadvantaged rural areas. The development of the CRISPR/Cas system in nucleic acid detection has allowed for pathogen point-of-care detection. Here, we developed a rapid and accurate point-of-care assay for HBV based on LAMP-Cas12a. It innovatively solves the problem of point-of-care testing in 10 min, particularly the problem of sample nucleic acid extraction. Based on LAMP-Cas12a, visualization of the assay results is presented by both a fluorescent readout and by lateral flow test strips. The lateral flow test strip technology can achieve results visible to the naked eye, while fluorescence readout can achieve real-time high-sensitivity detection. The fluorescent readout-based Cas12a assay can achieve HBV detection with a limit of detection of 1 copy/μL within 13 min, while the lateral flow test strip technique only takes 20 min. In the evaluation of 73 clinical samples, the sensitivity and specificity of both the fluorescence readout and lateral flow test strip method were 100%, and the results of the assay were fully comparable to qPCR. The LAMP-Cas12a-based HBV assay relies on minimal equipment to provide rapid, accurate test results and low costs, providing significant practical value for point-of-care HBV detection.
| S-EPMC8125043 | biostudies-literature
Project description:Detection of Burkholderia pseudomallei, a causative bacterium for melioidosis, remains a challenging undertaking due to long assay time, laboratory requirements, and the lack of specificity and sensitivity of many current assays. In this study, we are presenting a novel method that circumvents those issues by utilizing CRISPR-Cas12a coupled with isothermal amplification to identify B. pseudomallei DNA from clinical isolates. Through in silico search for conserved CRISPR-Cas12a target sites, we engineered the CRISPR-Cas12a to contain a highly specific spacer to B. pseudomallei, named crBP34. The crBP34-based detection assay can detect as few as 40 copies of B. pseudomallei genomic DNA while discriminating against other tested common pathogens. When coupled with a lateral flow dipstick, the assay readout can be simply performed without the loss of sensitivity and does not require expensive equipment. This crBP34-based detection assay provides high sensitivity, specificity and simple detection method for B. pseudomallei DNA. Direct use of this assay on clinical samples may require further optimization as these samples are complexed with high level of human DNA.
| S-EPMC9423629 | biostudies-literature
Project description:BackgroundMonkeypox (MPX), caused by the Monkeypox virus (MPXV), has incurred global attention since it broke out in many countries in recent times, which highlights the need for rapid and reliable diagnosis of MPXV infection.MethodsWe combined recombinase polymerase amplification (RPA) with CRISPR/Cas12a-based detection to devise a diagnostic test for detection of MPXV and differentiation of its two clades [Central Africa clade (MPXV-CA) and West Africa clade (MPXV-WA)], and called it MPXV-RCC. The sensitivity, specificity and practicability of this method have been analyzed.ResultsThe optimal conditions of MPXV-RCC assay include two RPA reactions at 38°C for 25 min and a CRISPR/Cas12a-gRNA detection at 37°C for 10 min. The results of MPXV-RCC assay were indicated by a real-time fluorescence analysis software. Thus, the whole detection process, including rapid template preparation (20 min), RPA reaction (25 min) and CRISPR-based detection (10 min), could be finished within 1 hour. The sensitivity of MPXV-RCC for MPXV-CA and MPXV-WA detection was down to 5~10 copies of recombination plasmids and pseudovirus per reaction. Particularly, MPXV-RCC assay could clearly differentiate MPXV-CA from MPXV-WA, and had no cross-reactivity with other pathogens. In addition, the feasibility of MPXV-RCC assay was further validated by using spiked clinical samples.ConclusionThe MPXV-RCC assay developed here is a promising tool for quick and reliable diagnosis of MPXV infection.
| S-EPMC10335395 | biostudies-literature
Project description:Cas12a-based systems, which detect specific nucleic acids via collateral cleavage of reporter DNA, display huge potentials for rapid diagnosis of infectious diseases. Here, the Manganese-enhanced Cas12a (MeCas12a) system is described, where manganese is used to increase the detection sensitivity up to 13-fold, enabling the detection of target RNAs as low as five copies. MeCas12a is also highly specific, and is able to distinguish between single nucleotide polymorphisms (SNPs) differing by a single nucleotide. MeCas12a can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples and distinguish between SARS-CoV-2 and Middle East respiratory syndrome coronavirus (MERS-CoV) RNA in simulated samples, thus offering an attractive alternative to other methods for the diagnosis of infectious diseases including COVID-19 and MERS.
| S-EPMC7536916 | biostudies-literature
Project description:Developing a strategy to specifically kill cancer cells without inducing obvious damage to normal cells may be of great clinical significance for cancer treatment. In the present study, we developed a new precise personalized strategy named "i-CRISPR" for cancer treatment through adding DNA damage repair inhibitors(i) and inducing cancer cell-specific DNA double strand breaks by CRISPR. Through in vitro and in vivo experiments, we confirmed the efficacy of this strategy in multiple cancer models and revealed the mechanism of cell death. Our strategy might provide a novel concept for precise cancer therapy.
| S-EPMC9380384 | 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:Aphelenchoides besseyi (A. besseyi), a seed-borne parasitic nematode, is the causal agent of rice white tip disease (RWTD), which may result in a drastic loss of rice yield. Seed treatments are currently considered to be the most effective means of preventing the spread of RWTD. Therefore, the rapid, highly specific, and accurate detection of A. besseyi from rice seeds is crucial for the surveillance, prevention, and control of RWTD. Here, we describe a novel detection assay that combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a to detect A. besseyi (termed RPA-Cas12a-Ab), with a low limit of detection (LOD) of 1 copy/μl of plasmid or 1:107 diluted DNA extracted from individual nematodes. To improve the user-friendliness, lateral flow strip assay (LFA) was adopted to visualize the detection result. The LOD of the RPA-Cas12a-Ab LFA assay was 1,000 copies/μl plasmid or 1:10 diluted DNA extracted from individual nematodes. The assay developed in this study was able to identify A. besseyi in 45 min with high accuracy and sensitivity without cross reaction with three closely related non-A. besseyi species. Thus, RPA-Cas12a-Ab is a rapid, sensitive, and specific detection system that requires no sophisticated equipment and shows promise for on-site surveillance of A. besseyi.
| S-EPMC9279656 | biostudies-literature
Project description:CRISPR-based detection of target DNA or RNA exploits a dual function, including target sequence-specific recognition followed by trans-cleavage activity of a collateral ssDNA linker between a fluorophore (F) and a quencher (Q), which amplifies a fluorescent signal upon cleavage. In this work, we have extended such dual functionality in a modified immunoassay format to detect a target protein, CXCL9, which is markedly elevated in the urine of kidney transplant recipients undergoing acute rejection episodes. To establish the "immuno-CRISPR" assay, we used anti-CXCL9 antibody-DNA barcode conjugates to target CXCL9 and amplify fluorescent signals via Cas12a-based trans-cleavage activity of FQ reporter substrates, respectively, and in the absence of an isothermal amplification step. To enhance detection sensitivity, the DNA barcode system was engineered by introducing multiple Cas12a recognition sites. Use of biotinylated DNA barcodes enabled self-assembly onto streptavidin (SA) to generate SA-DNA barcode complexes to increase the number and density of Cas12a recognition sites attached to biotinylated anti-CXCL9 antibody. As a result, we improved the rate of CXCL9 detection approximately 8-fold when compared to the use of a monomeric DNA barcode. The limit of detection (LOD) for CXCL9 using the immuno-CRISPR assay was 14 pg/mL, which represented an ∼7-fold improvement when compared to traditional HRP-based ELISA. Selectivity was shown with a lack of crossover reactivity with the related chemokine CXCL1. Finally, we successfully evaluated the presence of CXCL9 in urine samples from 11 kidney transplant recipients using the immuno-CRISPR assay, resulting in 100% accuracy to clinical CXCL9 determination and paving the way for use as a point-of-care noninvasive biomarker for the detection of kidney transplant rejection.
| S-EPMC9012118 | biostudies-literature