scholarly journals Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

2020 ◽  
Author(s):  
Long T. Nguyen ◽  
Brianna M. Smith ◽  
Piyush K. Jain
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Nucleic Acid Detection ◽  
Wild Type ◽  
Catalytic Behavior ◽  
Methylated Dna ◽  
Cleavage Activity ◽  
Target Dna ◽  
Optimized Conditions ◽  
Higher Sensitivity

AbstractThe CRISPR/Cas12a RNA-guided complexes have a tremendous potential for nucleic acid detection due to its ability to indiscriminately cleave ssDNA once bound to a target DNA. However, the current CRISPR/Cas12a systems are limited to detecting DNA in a picomolar detection limit without an amplification step. Here, we developed a platform with engineered crRNAs and optimized conditions that enabled us to detect DNA, DNA/RNA heteroduplex and methylated DNA with higher sensitivity, achieving a limit of detection of in femtomolar range without any target pre-amplification step. By extending the 3’- or 5’-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discovered a new self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA. We applied this sensitive system to detect as low as 25 fM dsDNA from the PCA3 gene, an overexpressed biomarker in prostate cancer patients, in simulated urine over 6 hours. The same platform was used to detect as low as ~700 fM cDNA from HIV, 290 fM RNA from HCV, and 370 fM cDNA from SARS-CoV-2, all within 30 minutes without a need for target amplification. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs were incorporated in a paper-based lateral flow assay that could detect the target with up to 23-fold higher sensitivity within 40-60 minutes.

scholarly journals Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Long T. Nguyen ◽  
Brianna M. Smith ◽  
Piyush K. Jain
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Divalent Cations ◽  
Nucleic Acid Detection ◽  
Wild Type ◽  
Detailed Characterization ◽  
Cleavage Activity ◽  
Optimized Conditions ◽  
Higher Sensitivity

AbstractThe CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the femtomolar range without any target pre-amplification step. By extending the 3′- or 5′-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discover a self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40–60 min.

scholarly journals Schnellnachweis von SARS-CoV-2 mit recombinase polymerase amplification

BIOspektrum ◽  
2020 ◽  
Vol 26 (6) ◽  
pp. 624-627
Author(s):  
Ole Behrmann ◽  
Iris Bachmann ◽  
Frank Hufert ◽  
Gregory Dame
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Dna Polymerase ◽  
Fluorescent Probes ◽  
Reverse Transcription ◽  
Nucleic Acid Detection ◽  
Detection Scheme ◽  
Recombination Proteins ◽  
Target Dna ◽  
Rapid Amplification

Abstract The COVID-19 pandemic highlights the need for fast and simple assays for nucleic acid detection. As an isothermal alternative to RT-qPCR, we outline the development of a detection scheme for SARS-CoV-2 RNA based on reverse transcription recombinase polymerase amplification (RT-RPA) technology. RPA uses recombination proteins in combination with a DNA polymerase for rapid amplification of target DNA at a constant temperature (39–42 °C) within 10 to 20 minutes and can be monitored in real-time with fluorescent probes.

scholarly journals 1832. Development of an Ultrasensitive Field-Applicable Plasmodium falciparum Assay for Malaria Diagnosis and Eradication

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S42-S43
Author(s):  
Rose Lee ◽  
Helena De Puig Guixe ◽  
Jeffrey Dvorin ◽  
James Collins
Keyword(s):  
Plasmodium Falciparum ◽  
Nucleic Acid ◽  
Genomic Dna ◽  
Limit Of Detection ◽  
Malaria Diagnosis ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
One Pot ◽  
Minimal Sample ◽  
Isothermal Assay

Abstract Background Malaria control and eradication have been hampered by asymptomatic carriage which serves as a parasite reservoir. Low-density infections (< 100 parasites/microliter) frequently fall below the limit of detection (LOD) of microscopy and rapid diagnostic tests (RDT) which are antigen-based tests. Molecular methods such as polymerase chain reaction are capable of higher sensitivity yet remain impractical for resource-limited settings. We describe development of an isothermal assay using the nucleic acid detection platform SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing), which may also be increasingly important as there has been rising detection of histidine-rich protein 2 (HRP2) gene deletions in Plasmodium spp. HRP2 is the most commonly used antigen in RDTs and deletion of this gene would render many RDTs obsolete. Methods SHERLOCK leverages the endonucleases of CRISPR-associated microbial adaptive immunity. Cas12a is an RNA-guided, DNA-cleaving enzyme, which can be programmed with guide RNAs to cleave nontarget reporter ssDNA. We exploit the nonspecific degradation of labeled ssDNA to detect the presence of the dsDNA target that activated Cas12a (Figure 1). Recombinase polymerase amplification (RPA) coupled with Cas12a detection enables a lower LOD. Plasmodium falciparum whole genomic DNA was compared with parasites cultured in red blood cells (RBCs) with known parasitemia and boiled at 95°C for 5 minutes for lysis of RBCs/parasites then diluted 1:2.5 to prevent solidification. Results This SHERLOCK assay detected simulated Plasmodium falciparum infection at attomolar LODs when applied to whole genomic DNA and simulated samples of infected RBCs spiked into whole blood. The genomic assay detected down to 0.2 parasites/microliter and the simulated sample detected to 10 parasites/microliter in the final reaction volume. In comparison, LODs from the initial input volume was 5aM and 250aM, respectively (Figure 2). Conclusion We demonstrate an isothermal nucleic acid detection platform capable of diagnosis in 60 minutes in a one-pot assay requiring minimal sample preparation and reaching an LOD recommended by the WHO for malaria eradication. In summary, we illustrate the utility of the SHERLOCK platform in application to malaria and global health. Disclosures All Authors: No reported Disclosures.

Colorimetric Nucleic Acid Detection Based on Gold Nanoparticles with Branched DNA

NANO ◽  
2020 ◽  
Vol 15 (08) ◽  
pp. 2050110
Author(s):  
Zhikun Zhang ◽  
Xiaojie Ye ◽  
Qingqing Liu ◽  
Cuixia Hu ◽  
Jimmy Yun ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Nucleic Acid ◽  
Genetic Diseases ◽  
Colorimetric Detection ◽  
Nucleic Acid Detection ◽  
Great Promise ◽  
Dna Nanostructures ◽  
Practical Applications ◽  
Branched Dna ◽  
Target Dna

Nucleic acid detection is becoming increasingly important in the diagnostics of genetic diseases for biological analysis. We herein propose gold nanoparticles as probe for colorimetric detection of nucleic acids with branched DNA nanostructures, which enables a novel and simple colorimetric biosensor. In our system, the target DNA specifically triggered two short-chain ssDNA probes to generate branched DNA nanostructures (Y-shape DNA), which prevent AuNPs from aggregation in aqueous NaCl solution. On the contrary, when the target DNA did not exist, gold nanoparticles were unstable and aggregated easily because there is no anti-aggregation function from Y-shape DNA. Sensor response was found to be proportional to the target DNA concentration from 5 to 100[Formula: see text]nM, with detection limits determined as 5[Formula: see text]nM. The developed platform is for colorimetric nucleic acid detection without enzymes, label and modification holds great promise for practical applications.

scholarly journals One-Tube SARS-CoV-2 Detection Platform Based on RT-RPA and CRISPR/Cas12a

2020 ◽  
Author(s):  
Yangyang Sun ◽  
Lei Yu ◽  
Chengxi Liu ◽  
Wei Chen ◽  
Dechang Li ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
N Gene ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Detection Process ◽  
Human Coronaviruses ◽  
Negative Controls

Abstract Background: COVID-19 has spread rapidly around the world, affecting almost every person. 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. Methods: We designed RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) primers of RdRp gene and N gene according to the SARS-CoV-2 gene sequence. We optimized the components in the reaction so that the detection process could be carried out in one tube. The specificity was demonstrated through detecting nucleic acid samples from seven human coronaviruses. Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards diluted by different gradients were used to demonstrate the limit of detection. Furthermore, we have developed a lateral flow assay based on OR-DETECTR for the detection of COVID-19. Results: We have developed a o ne-tube detection platform based on R T- R PA and DNA Endonuclease-Targeted CRISPR Trans Reporter ( DETECTR ) technology, termed OR-DETECTR, to detect SARS-CoV-2. The detection process is completed in one tube, and the time is 50min. The method can specifically detect SARS-CoV-2 from seven human coronaviruses with a low detection limit of 2.5 copies/µl input. Results from six SARS-CoV-2 patient samples, 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. Furthermore, we have developed a lateral flow assay based on OR-DETECTR for the detection of COVID-19. Conclusions: OR-DETECTR detection platform is rapid, accurate, tube closed, easy-to-operate, and free of large instruments for COVID-19 detection.

An autocatalytic DNA machine with autonomous target recycling and cascade circular exponential amplification for one-pot, isothermal and ultrasensitive nucleic acid detection

2016 ◽  
Vol 52 (74) ◽  
pp. 11108-11111 ◽  
Author(s):  
Xinya Sun ◽  
Li Wang ◽  
Mingsha Zhao ◽  
Changzhi Zhao ◽  
Shufeng Liu
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Detection ◽  
Ultrasensitive Detection ◽  
One Pot ◽  
Target Recycling ◽  
Target Dna ◽  
Dna Machine ◽  
The One ◽  
Amplification Strategy

An autonomous target recycling and cascade circular exponential amplification strategy was proposed for the one-pot, isothermal and ultrasensitive detection of target DNA.

scholarly journals Optimization of a Nucleic Acid-Based Reporter System To Detect Mycobacterium tuberculosis Antibiotic Sensitivity

2014 ◽  
Vol 59 (1) ◽  
pp. 407-413 ◽  
Author(s):  
Matthew C. Mulvey ◽  
Margaret Lemmon ◽  
Stephanie Rotter ◽  
Jonathan Lees ◽  
Leo Einck ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nucleic Acid ◽  
Limit Of Detection ◽  
Surrogate Marker ◽  
Antibiotic Sensitivity ◽  
Nucleic Acid Detection ◽  
Drug Resistant ◽  
Reporter System ◽  
Susceptible Population ◽  
Content Type

ABSTRACTWe previously reported the development of a prototype antibiotic sensitivity assay to detect drug-resistantMycobacterium tuberculosisusing infection by mycobacteriophage to create a novel nucleic acid transcript, a surrogate marker of mycobacterial viability, detected by reverse transcriptase PCR (M. C. Mulvey et al., mBio3:e00312-11, 2012). This assay detects antibiotic resistance to all drugs, even drugs for which the resistance mechanism is unknown or complex: it is a phenotypic readout using nucleic acid detection. In this report, we describe development and characteristics of an optimized reporter system that directed expression of the RNA cyclase ribozyme, which generated circular RNA through an intramolecular splicing reaction and led to accumulation of a new nucleic acid sequence in phage-infected bacteria. These modifications simplified the assay, increased the limit of detection from 104to <102M. tuberculosiscells, and correctly identified the susceptibility profile ofM. tuberculosisstrains exposed for 16 h to either first-line or second-line antitubercular drugs. In addition to phenotypic drug resistance or susceptibility, the assay reported streptomycin MICs and clearly detected 10% drug-resistant cells in an otherwise drug-susceptible population.

scholarly journals A low-cost fluorescence reader for in vitro transcription and nucleic acid detection with Cas13a

2019 ◽  
Author(s):  
Florian Katzmeier ◽  
Lukas Aufinger ◽  
Aurore Dupin ◽  
Jorge Quinteiro ◽  
Matthias Lenz ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Point Of Care ◽  
Low Cost ◽  
Nucleic Acid Detection ◽  
Fluorescence Detector ◽  
Flat Detector ◽  
Low Resource Settings ◽  
Low Resource

AbstractPoint-of-care testing (POCT) in low-resource settings requires tools that can operate independent of typical laboratory infrastructure. Due to its favorable signal-to-background ratio, a wide variety of biomedical tests utilize fluorescence as a readout. However, fluorescence techniques often require expensive or complex instrumentation and can be difficult to adapt for POCT. To address this issue, we developed a pocket-sized fluorescence detector costing less than $15 that is easy to manufacture and can operate in low-resource settings. It is built from standard electronic components, including an LED and a light dependent resistor, filter foils and 3D printed parts, and reliably detected less than 10 nM fluorescein concentrations (with a lower limit of detection of ≈6.8 nM), which is sufficient to follow typical biochemical reactions used in POCT applications. All assays are conducted on filter paper, which allows for a flat detector architecture to improve signal collection. We validate the device by quantifying in vitro RNA transcription and also demonstrate sequence-specific detection of target RNAs in the nanomolar range using a Cas13a-based fluorescence assay. Cas13a is a RNA-guided, RNA-targeting CRISPR effector with promiscuous RNase activity upon recognition of its RNA target. Cas13a sensing is highly specific and adaptable and in combination with our detector represents a promising approach for nucleic acid POCT. Furthermore, our open-source device architecture could be a valuable educational tool that integrates hardware, software and biochemistry concepts.

scholarly journals Author Correction: Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Long T. Nguyen ◽  
Brianna M. Smith ◽  
Piyush K. Jain
Keyword(s):  
Nucleic Acid ◽  
Nucleic Acid Detection ◽  
Cleavage Activity

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-20117-z

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