Project description:There is a need for point-of-care bacterial sensing and identification technologies that are rapid and simple to operate. Technologies that do not rely on growth cultures, nucleic acid amplification, step-wise reagent addition, and complex sample processing are the key for meeting this need. Herein, multiple materials technologies are integrated for overcoming the obstacles in creating rapid and one-pot bacterial sensing platforms. Liquid-infused nanoelectrodes are developed for reducing nonspecific binding on the transducer surface; bacterium-specific RNA-cleaving DNAzymes are used for bacterial identification; and redox DNA barcodes embedded into DNAzymes are used for binding-induced electrochemical signal transduction. The resultant single-step and one-pot assay demonstrates a limit-of-detection of 102 CFU mL-1 , with high specificity in identifying Escherichia coli amongst other Gram positive and negative bacteria including Klebsiella pneumoniae, Staphylococcus aureus, and Bacillus subtilis. Additionally, this assay is evaluated for analyzing 31 clinically obtained urine samples, demonstrating a clinical sensitivity of 100% and specify of 100%. When challenging this assay with nine clinical blood cultures, E. coli-positive and E. coli-negative samples can be distinguished with a probability of p < 0.001.
Project description:The one-pot synthesis of a target molecule in the same reaction vessel is widely considered to be an efficient approach in synthetic organic chemistry. In this review, the characteristics and limitations of various one-pot syntheses of biologically active molecules are explained, primarily involving organocatalytic methods as key tactics. Besides catalysis, the pot-economy concepts presented herein are also applicable to organometallic and organic reaction methods in general.
Project description:CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clinically in 80 serum samples for hepatitis C virus (HCV) and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and accuracy. We obtain results in as little as 20 min, and with the extraction process, the entire assay can be performed within an hour.
Project description:Data files and supplementary data associated with the one-pot method. These files are the results of one-pot enrichment of both acetylated and succinylated peptides.
Project description:CRISPR-Cas12a based one-pot detection system has been used in nucleic acid detection and diagnosis. However, it is not sensitive enough to distinguish single nucleotide polymorphisms (SNP), which has greatly restricted its application. To overcome these limitations, we engineered a LbCas12a variant with enhanced sensitivity against SNP, named seCas12a (sensitive Cas12a). SeCas12a-based one-pot SNP detection system is a versatile platform that could use both canonical and non-canonical PAM, and was almost not limited by mutation types to distinguish SNPs located between position 1 to 17. The use of truncated crRNA further improved SNP specificity of seCas12a. Mechanistically, we found only when the cis-cleavage was at low level between 0.01min-1 and 0.0006 min-1, a good signal-to-noise ratio can be achieved in one-pot test. SeCas12a-based one-pot SNP detection system was applied to detect pharmacogenomic SNPs in human clinical samples. Of thirteen donors tested in two different SNPs, the seCas12a mediated one-pot system could faithfully detect the SNPs in 30 min with 100% accuracy.
Project description:We reported a one-pot fluorescence-based assay to quantitively detect A3A activity combined with cytosine deamination and uracil excision. After deamination by A3A and USER enzyme treatment, the fluorescent turn-on effect at 520 nm was observed, which can be used to evaluate the A3A activity and screen inhibitors.
Project description:MicroRNAs (miRNAs) and short RNA fragments (18-25 nt) are crucial biomarkers in biological research and disease diagnostics. However, their accurate and rapid detection remains a challenge, largely due to their low abundance, short length, and sequence similarities. In this study, we report on a highly sensitive, one-step RNA O-circle amplification (ROA) assay for rapid and accurate miRNA detection. The ROA assay commences with the hybridization of a circular probe with the test RNA, followed by a linear rolling circle amplification (RCA) using dUTP. This amplification process is facilitated by U-nick reactions, which lead to an exponential amplification for readout. Under optimized conditions, assays can be completed within an hour, producing an amplification yield up to the microgram level, with a detection limit as low as 0.15 fmol (6 pM). Notably, the ROA assay requires only one step, and the results can be easily read visually, making it user-friendly. This ROA assay has proven effective in detecting various miRNAs and phage ssRNA. Overall, the ROA assay offers a user-friendly, rapid, and accurate solution for miRNA detection.Supplementary informationThe online version contains supplementary material available at 10.1007/s42994-024-00140-0.
Project description:IntroductionThe ongoing 2019 coronavirus disease pandemic (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, is a global public health threat. Early diagnosis and identification of SARS-CoV-2 and its variants plays a critical role in COVID-19 prevention and control. Currently, the most widely used technique to detect SARS-CoV-2 is quantitative reverse transcription real-time quantitative PCR (RT-qPCR), which takes nearly 1 hour and should be performed by experienced personnel to ensure the accuracy of results. Therefore, the development of a nucleic acid detection kit with higher sensitivity, faster detection and greater accuracy is important.MethodsHere, we optimized the system components and reaction conditions of our previous detection approach by using RT-RAA and Cas12b.ResultsWe developed a Cas12b-assisted one-pot detection platform (CDetection.v2) that allows rapid detection of SARS-CoV-2 in 30 minutes. This platform was able to detect up to 5,000 copies/ml of SARS-CoV-2 without cross-reactivity with other viruses. Moreover, the sensitivity of this CRISPR system was comparable to that of RT-qPCR when tested on 120 clinical samples.DiscussionThe CDetection.v2 provides a novel one-pot detection approach based on the integration of RT-RAA and CRISPR/Cas12b for detecting SARS-CoV-2 and screening of large-scale clinical samples, offering a more efficient strategy for detecting various types of viruses.
Project description:African swine fever virus (ASFV) is a leading cause of worldwide agricultural loss. ASFV is a highly contagious and lethal disease for both domestic and wild pigs, which has brought enormous economic losses to a number of countries. Conventional methods, such as general polymerase chain reaction and isothermal amplification, are time-consuming, instrument-dependent, and unsatisfactorily accurate. Therefore, rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control. Herein, we created a one-pot visual detection system for ASFV with CRISPR/Cas12a technology combined with LAMP or RPA. A mineral oil sealing strategy was adopted to mitigate sample cross-contamination between parallel vials during high-throughput testing. Furthermore, the blue fluorescence signal produced by ssDNA reporter could be observed by the naked eye without any dedicated instrument. For CRISPR-RPA system, detection could be completed within 40 min with advantageous sensitivity. While CRISPR-LAMP system could complete it within 60 min with a high sensitivity of 5.8 × 102 copies/μl. Furthermore, we verified such detection platforms display no cross-reactivity with other porcine DNA or RNA viruses. Both CRISPR-RPA and CRISPR-LAMP systems permit highly rapid, sensitive, specific, and low-cost Cas12a-mediated visual diagnostic of ASFV for point-of-care testing (POCT) applications.