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.

scholarly journals Scanning single-molecule counting system for Eprobe with highly simple and effective approach

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243319
Author(s):  
Takeshi Hanami ◽  
Tetsuya Tanabe ◽  
Takuya Hanashi ◽  
Mitsushiro Yamaguchi ◽  
Hidetaka Nakata ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Detection System ◽  
Signal To Noise Ratio ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Digital Measurement ◽  
Excitation Light ◽  
Target Nucleic Acid ◽  
Fluorescent Molecules

Here, we report a rapid and ultra-sensitive detection technique for fluorescent molecules called scanning single molecular counting (SSMC). The method uses a fluorescence-based digital measurement system to count single molecules in a solution. In this technique, noise is reduced by conforming the signal shape to the intensity distribution of the excitation light via a circular scan of the confocal region. This simple technique allows the fluorescent molecules to freely diffuse into the solution through the confocal region and be counted one by one and does not require statistical analysis. Using this technique, 28 to 62 aM fluorescent dye was detected through measurement for 600 s. Furthermore, we achieved a good signal-to-noise ratio (S/N = 2326) under the condition of 100 pM target nucleic acid by only mixing a hybridization-sensitive fluorescent probe, called Eprobe, into the target oligonucleotide solution. Combination of SSMC and Eprobe provides a simple, rapid, amplification-free, and high-sensitive target nucleic acid detection system. This method is promising for future applications to detect particularly difficult to design primers for amplification as miRNAs and other short oligo nucleotide biomarkers by only hybridization with high sensitivity.

Amplification-Free DNA Detection Using a Microtip-Sensor Decorated With LNA Probes for Rapid TB Screening

2011 ◽  
Author(s):  
Shinnosuke Inoue ◽  
Woon-Hong Yeo ◽  
Jong-Hoon Kim ◽  
Jae-Hyun Chung ◽  
Kyong-Hoon Lee ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Staphylococcus Epidermidis ◽  
Genomic Dna ◽  
Bacterial Culture ◽  
Low Cost ◽  
Surrogate Marker ◽  
High Sensitivity ◽  
Diagnostic Methods ◽  
Nucleic Acid Amplification ◽  
Highly Sensitive

Tuberculosis (TB) is an epidemic affecting one-third of the world’s population, mostly in developing and low-resource settings. People having active pulmonary TB are considered highly infectious; therefore, it is critical to identify and treat these patients rapidly before spreading to others. However, the most reliable TB diagnostic methods of bacterial culture or nucleic acid amplification are time-consuming and expensive. The challenge of TB diagnosis lies in highly sensitive and specific screening with low cost. Here, we present an LNA-modified microtip-sensor, which is capable of selectively detecting low-abundance DNA from bacteria. When genomic DNA of Bacillus Calmette-Gue´rin (BCG, a surrogate marker of Mycobacterium bovis), and genomic DNA of Staphylococcus epidermidis (S. epi) are used, the microtip-sensor yields the detection limit of 1,000 copies/mL within 20 minutes. The high sensitivity and specificity approaching nucleic acid amplification methods can potentially overcome the current challenges for rapid TB screening.

Exploring 'best practice' for nucleic acid detection of Neisseria gonorrhoeae

Sexual Health ◽  
2008 ◽  
Vol 5 (1) ◽  
pp. 17 ◽  
Author(s):  
David M. Whiley ◽  
Suzanne M. Garland ◽  
Geoffrey Harnett ◽  
Gary Lum ◽  
David W. Smith ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Neisseria Gonorrhoeae ◽  
Best Practice ◽  
Current Knowledge ◽  
False Negative ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Predictive Values ◽  
Neisseria Species ◽  
Negative Results

Nucleic acid detection tests (NADT) have considerable benefits for the detection of Neisseria gonorrhoeae (GC), including high sensitivity across a range of specimen types and use under widely differing settings and conditions. However, sexual health practitioners and others who use data generated by NADT for GC should be aware of some important limitations of these tests. False-positive results caused by cross reaction with commensal Neisseria species have been observed in many assays, and have lead to unacceptably low positive-predictive values in some patient populations. Further, false-negative results can be caused by GC sequence variation, with some gonococci lacking certain NADT target sequences. This review examines the issues associated with gonococcal NADT and considers best practice for use of these assays based on current knowledge. We emphasise the need for supplementary testing and extensive assay validation, and suggest appropriate strategies for these requirements irrespective of the setting in which they are used. Further, we highlight the need to maintain culture-based testing for certain specimen sites as well as for antimicrobial resistance surveillance.

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.

scholarly journals Virus detection via programmable Type III-A CRISPR-Cas systems

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sagar Sridhara ◽  
Hemant N. Goswami ◽  
Charlisa Whyms ◽  
Jonathan H. Dennis ◽  
Hong Li
Keyword(s):  
Nucleic Acid ◽  
Detection Method ◽  
Virus Detection ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Immune Effector ◽  
Type Iii ◽  
System A ◽  
Acid Cleavage

AbstractAmong the currently available virus detection assays, those based on the programmable CRISPR-Cas enzymes have the advantage of rapid reporting and high sensitivity without the requirement of thermocyclers. Type III-A CRISPR-Cas system is a multi-component and multipronged immune effector, activated by viral RNA that previously has not been repurposed for disease detection owing in part to the complex enzyme reconstitution process and functionality. Here, we describe the construction and application of a virus detection method, based on an in vivo-reconstituted Type III-A CRISPR-Cas system. This system harnesses both RNA- and transcription-activated dual nucleic acid cleavage activities as well as internal signal amplification that allow virus detection with high sensitivity and at multiple settings. We demonstrate the use of the Type III-A system-based method in detection of SARS-CoV-2 that reached 2000 copies/μl sensitivity in amplification-free and 60 copies/μl sensitivity via isothermal amplification within 30 min and diagnosed SARS-CoV-2-infected patients in both settings. The high sensitivity, flexible reaction conditions, and the small molecular-driven amplification make the Type III-A system a potentially unique nucleic acid detection method with broad applications.

scholarly journals Nucleic acid detection using a universal electrochemical sensor for SNS differentiation

2018 ◽  
pp. 79-86
Keyword(s):  
Nucleic Acid ◽  
Electrochemical Sensor ◽  
Nucleotide Substitution ◽  
High Sensitivity ◽  
Biomedical Science ◽  
Nucleic Acid Detection ◽  
Single Nucleotide Substitution ◽  
University Of Central Florida ◽  
Single Nucleotide ◽  
Central Florida

Nucleic acid detection using a universal electrochemical sensor for SNS differentiation Detección de ácidos nucleicos usando un sensor electroquímico universal para la discriminación de SNS Percy Calvo-Marzal1, Dmitry M. Kolpashchikov1,2,3 and Karin Y. Chumbimuni-Torres1 1 Department of Chemistry, University of Central Florida, 4000 Central Florida Blvd., Orlando, FL 32816 2 National Center for Forensic Science, University of Central Florida, Orlando, FL 32816 3 Burnett School of Biomedical Science, University of Central Florida, Orlando, FL 32816 Recibido el 26 de diciembre del 2017, aceptado el 31 de diciembre del 2017 DOI: https://doi.org/10.33017/RevECIPeru2017.0008/ Abstract Nucleic acid detection with high sensitivity and selectivity capabilities could aid in the diagnosis of varies diseases such as: infections, cancer, among others. Discrimination of a mismatch or single nucleotide substitution (SNS) is challenge due to a minimum pair to pair formation double strand needed to stabilize the structure. Here we present a new approach to atinge the needed stability by using additional segments strains to favored stabilization. The use of additional complementary strains facilitates the discrimination of a SNS in a cooperative way. Keywords: Nuclei acid, Single nucleotide substitution, infection, cancer

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.

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 PfHRP2 detection using plasmonic optrodes: performance analysis

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Médéric Loyez ◽  
Mathilde Wells ◽  
Stéphanie Hambÿe ◽  
François Hubinon ◽  
Bertrand Blankert ◽  
...  
Keyword(s):  
Optical Fiber ◽  
Limit Of Detection ◽  
Fiber Surface ◽  
Malaria Diagnosis ◽  
High Sensitivity ◽  
Cost Effective ◽  
Label Free ◽  
Multiple Receptors ◽  
Label Free Detection

Abstract Background Early malaria diagnosis and its profiling require the development of new sensing platforms enabling rapid and early analysis of parasites in blood or saliva, aside the widespread rapid diagnostic tests (RDTs). Methods This study shows the performance of a cost-effective optical fiber-based solution to target the presence of Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Unclad multimode optical fiber probes are coated with a thin gold film to excite Surface Plasmon Resonance (SPR) yielding high sensitivity to bio-interactions between targets and bioreceptors grafted on the metal surface. Results Their performances are presented in laboratory conditions using PBS spiked with growing concentrations of purified target proteins and within in vitro cultures. Two probe configurations are studied through label-free detection and amplification using secondary antibodies to show the possibility to lower the intrisic limit of detection. Conclusions As malaria hits millions of people worldwide, the improvement and multiplexing of this optical fiber technique can be of great interest, especially for a future purpose of using multiple receptors on the fiber surface or several coated-nanoparticles as amplifiers.

Sign in / Sign up

Export Citation Format

Share Document