ADESSO detects SARS-CoV-2 and its variants: extensive clinical validation of an optimised CRISPR-Cas13-based COVID-19 test

With the coronavirus disease 19 (COVID-19) pandemic now deep into its second year, widespread testing for the detection of the causative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is fundamental. The gold standard reverse transcription quantitative PCR (RT-qPCR) cannot keep up with the high demand alone, therefore alternative diagnostic tests are needed. Here we present ADESSO (Accurate Detection of Evolving SARS-CoV-2 through SHERLOCK Optimisation), an optimised version of the CRISPR-based SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) assay. After an extensive validation on 983 clinical samples, we demonstrated that ADESSO has a sensitivity of 96% and a specificity of 100% on extracted RNA, comparable to RT-qPCR. Its performance on unextracted samples still allows the detection of the more infectious 75% of the COVID-19 positive population, making it suitable for point-of-care (POC) testing. Interestingly, our in parallel comparison of 390 matching swab and gargle samples showed consistently lower viral loads in gargle specimens. We also validated ADESSO for the detection of the B.1.1.7 variant and demonstrated that ADESSO is adaptable to any variant of concern in less than one week, a critical feature now that worrisome SARS-CoV-2 variants are spreading all around the world.

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Ultrarapid, Ultrasensitive One-Step Kinetic Immunoassay for C-Reactive Protein (CRP) in Whole Blood Samples: Measurement of the Entire CRP Concentration Range with a Single Sample Dilution

Clinical Chemistry ◽

10.1093/clinchem/48.2.269 ◽

2002 ◽

Vol 48 (2) ◽

pp. 269-277 ◽

Cited By ~ 12

Author(s):

Piia Tarkkinen ◽

Tom Palenius ◽

Timo Lövgren

Keyword(s):

Whole Blood ◽

Solid Phase ◽

Point Of Care ◽

High Sensitivity ◽

Clinical Samples ◽

Cardiac Complications ◽

Fluorescence Signal ◽

C Reactive Protein ◽

Reactive Protein ◽

One Step

Abstract Background: Recently, measurement of very low concentrations of C-reactive protein (CRP) has gained popularity as a potential new means for predicting the risk of future cardiac complications. In this study, we demonstrate the feasibility of a kinetic, one-step microparticle assay for quantitative determination of extremely low and high CRP concentrations in the limited timeframe typical for point-of-care testing. Methods: A noncompetitive, kinetic CRP immunoassay was developed that uses individual, porous microparticles as the solid phase. The microparticles were covalently coated with a monoclonal capture antibody, and the monoclonal detection antibody was labeled with europium. The one-step binding reaction was stopped by washing after 2 min of incubation, and the fluorescence signal of individual particles was measured. Results: The analytical detection limit (mean of zero calibrator + 3 SD) was 0.00016 mg/L CRP. Clinical samples were diluted 400-fold before assay to cover the CRP concentration range of 0.064–1200 mg/L. The assay correlated well with the Dade Behring N High Sensitivity CRP assay (for 0–10 mg/L, r = 0.969, Sy|x = 0.68, n = 54; for 0–350 mg/L, r = 0.969, Sy|x = 11.7, n = 100). The within- and between-run CVs based on calculated concentrations were, respectively, 9–16% and 14% at 0.11 mg/L, 4.5–12% and 8.2% at 4.2 mg/L, and 3.5–6.3% and 4.4% at 105 mg/L, with a CV <15% at 0.2 mg/L and above. Conclusions: Use of the kinetic microparticle approach combined with time-resolved fluorometry allows ultrasensitive quantification of CRP in whole blood in 2 min with a linear assay range spanning more than four orders of magnitude.

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Toward point-of-care diagnostics of Candida auris

10.1101/2020.02.10.20021683 ◽

2020 ◽

Cited By ~ 1

Author(s):

Geoffrey Mulberry ◽

Sudha Chaturvedi ◽

Vishnu Chaturvedi ◽

Brian N. Kim

Keyword(s):

New York ◽

Real Time ◽

Real Time Pcr ◽

Positive Impact ◽

Point Of Care ◽

Low Cost ◽

High Sensitivity ◽

Clinical Samples ◽

Pcr Assay ◽

Candida Auris

AbstractCandida auris is a multidrug-resistant yeast that presents global health threat for the hospitalized patients. Early diagnostic of C. auris is crucial in control, prevention, and treatment. Candida auris is difficult to identify with standard laboratory methods and often can be misidentified leading to inappropriate management. A newly-devised real-time PCR assay played an important role in the ongoing investigation of the C. auris outbreak in New York metropolitan area. The assay can rapidly detect C. auris DNA in surveillance and clinical samples with high sensitivity and specificity, and also useful for confirmation of C. auris cultures. Despite its positive impact, the real-time PCR assay is difficult to deploy at frontline laboratories due to high-complexity set-up and operation. Using a low-cost handheld real-time PCR device, we show that the C. auris can potentially be identified in a low-complexity assay without the need for high-cost equipment. An implementation of low-cost real-time PCR device in hospitals and healthcare facilities is likely to accelerate the diagnosis of C. auris and for control of the global epidemic.

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Colorimetric RT-LAMP SARS-CoV-2 diagnostic sensitivity relies on color interpretation and viral load

Scientific Reports ◽

10.1038/s41598-021-88506-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Mateus Nóbrega Aoki ◽

Bruna de Oliveira Coelho ◽

Luiz Gustavo Bentim Góes ◽

Paola Minoprio ◽

Edison Luiz Durigon ◽

...

Keyword(s):

Viral Load ◽

Reaction Time ◽

Color Change ◽

Rna Virus ◽

Point Of Care ◽

Respiratory Viruses ◽

Lamp Reaction ◽

Clinical Samples ◽

Viral Loads ◽

Naked Eye

AbstractThe use of RT-LAMP (reverse transcriptase—loop mediated isothermal amplification) has been considered as a promising point-of-care method to diagnose COVID-19. In this manuscript we show that the RT-LAMP reaction has a sensitivity of only 200 RNA virus copies, with a color change from pink to yellow occurring in 100% of the 62 clinical samples tested positive by RT-qPCR. We also demonstrated that this reaction is 100% specific for SARS-CoV-2 after testing 57 clinical samples infected with dozens of different respiratory viruses and 74 individuals without any viral infection. Although the majority of manuscripts recently published using this technique describe only the presence of two-color states (pink = negative and yellow = positive), we verified by naked-eye and absorbance measurements that there is an evident third color cluster (orange), in general related to positive samples with low viral loads, but which cannot be defined as positive or negative by the naked eye. Orange colors should be repeated or tested by RT-qPCR to avoid a false diagnostic. RT-LAMP is therefore very reliable for samples with a RT-qPCR Ct < 30 being as sensitive and specific as a RT-qPCR test. All reactions were performed in 30 min at 65 °C. The use of reaction time longer than 30 min is also not recommended since nonspecific amplifications may cause false positives.

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CRISPR-cas13 enzymology rapidly detects SARS-CoV-2 fragments in a clinical setting

10.1101/2020.12.17.20228593 ◽

2020 ◽

Author(s):

Wahab A. Khan ◽

Rachael E. Barney ◽

Gregory J. Tsongalis

Keyword(s):

Clinical Setting ◽

High Sensitivity ◽

Limited Resources ◽

N Gene ◽

Clinical Samples ◽

Nucleic Acid Detection ◽

Viral Loads ◽

New Thinking ◽

And Performance ◽

Selection Of

ABSTRACTThe well-recognized genome editing ability of the CRISPR-Cas system has triggered significant advances in CRISPR diagnostics. This has prompted an interest in developing new biosensing applications for nucleic acid detection. Recently, such applications have been engineered for detection of SARS-CoV-2. Increased demand for testing and consumables of RT-qPCR assays has led to the use of alternate testing options in some cases. Here we evaluate the accuracy and performance of a novel fluorescence based assay that received EUA authorization from the FDA for detecting SARS-CoV-2 in clinical samples. The Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology forms the basis of the Sherlock CRISPR SARS-CoV-2 kit using the CRISPR-Cas13a system. Our experimental strategy included selection of COVID-19 patient samples from previously validated RT-qPCR assays. Positive samples were selected based on a broad range of cycle thresholds. A total of 50 COVID-19 patient samples were correctly diagnosed with 100% accuracy (relative fluorescence ratios: N gene 95% CI 23.2-36.3, ORF1ab gene 95% CI 27.6-45.4). All controls, including RNase P, showed expected findings. Overall ratios were robustly distinct between positive and negative cases relative to the pre-established 5-fold change in fluorescence read output. We have evaluated the accuracy of detecting conserved targets of SARS-CoV-2 across a range of viral loads using the SHERLOCK CRISPR collateral detection reaction in a clinical setting. These findings demonstrate encouraging results, especially at a time when COVID-19 clinical diagnosis is in high demand; often with limited resources. This approach highlights new thinking in infectious disease identification and can be expanded to measure nucleic acids in other clinical isolates.

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Rapid, accurate, nucleobase detection using FnCas9

10.1101/2020.09.13.20193581 ◽

2020 ◽

Cited By ~ 2

Author(s):

Mohd. Azhar ◽

Rhythm Phutela ◽

Manoj Kumar ◽

Asgar Hussain Ansari ◽

Riya Rauthan ◽

...

Keyword(s):

Point Of Care ◽

Disease Outbreaks ◽

Smartphone Application ◽

Clinical Samples ◽

Single Nucleotide Variants ◽

Clinical Prognosis ◽

Viral Loads ◽

Infectious Disease Outbreaks ◽

Dna And Rna ◽

True Outcome

Rapid detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for accelerated clinical prognosis as has been witnessed during the recent COVID-19 outbreak. Traditional methods relying on qPCR or sequencing are difficult to implement in settings with limited resources necessitating the development of accurate alternative testing strategies that perform robustly. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a direct Cas9 based enzymatic readout for detecting nucleotide sequences and identifying nucleobase identity without the requirement of trans-cleavage activity of reporter molecules. We demonstrate that FELUDA is 100% accurate in detecting single nucleotide variants (SNVs) including heterozygous carriers of a mutation and present a simple design strategy in the form of a web-tool, JATAYU, for its implementation. FELUDA is semi quantitative, can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications such as infectious disease outbreaks like COVID-19. Using a lateral flow readout within 1h, FELUDA shows 100% sensitivity and 97% specificity across all range of viral loads in clinical samples. In combination with RT-RPA and a smartphone application True Outcome Predicted via Strip Evaluation (TOPSE), we present a prototype for FELUDA for CoV-2 detection at home.

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Nanomonitors: Electrical Immunoassays for Protein Biomarker Profiling

MRS Proceedings ◽

10.1557/proc-1106-pp03-02 ◽

2008 ◽

Vol 1106 ◽

Author(s):

Manish Bothara ◽

Ravi K Reddy ◽

Thomas Barrett ◽

John Carruthers ◽

Shalini Prasad

Keyword(s):

Microelectrode Array ◽

Performance Metrics ◽

Point Of Care ◽

High Sensitivity ◽

Disease Diagnosis ◽

Clinical Samples ◽

Label Free ◽

Early Disease ◽

Alumina Membrane ◽

Protein Biomarker

AbstractThe objective of this research is to develop a “point-of-care” device for early disease diagnosis through protein biomarker characterization. Here we present label-free, high sensitivity detection of proteins with the use of electrical immunoassays that we call Nanomonitors. The basis of the detection principle lies in the formation of an electrical double layer and its perturbations caused by proteins trapped in a nanoporous alumina membrane over a microelectrode array platform. High sensitivity and rapid detection of two inflammatory biomarkers, C-reactive protein (CRP) and Myeloperoxidase (MPO) in pure and clinical samples through label-free electrical detection were achieved. The performance metrics achieved by this device makes it suitable as a “lab-on-a-chip” device for protein biomarker profiling and hence early disease diagnosis.

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Microfluidics-Enabled Digital Isothermal Cas13a Assay

10.1101/2021.08.18.21262201 ◽

2021 ◽

Author(s):

Frank X. Liu ◽

Johnson Q. Cui ◽

Hojeong Park ◽

Ka Wai Chan ◽

Tyler Leung ◽

...

Keyword(s):

Point Of Care ◽

Absolute Quantification ◽

Droplet Microfluidics ◽

Clinical Samples ◽

Quantitative Detection ◽

One Pot ◽

Viral Loads ◽

Assay Optimization ◽

Point Of Care Diagnostics ◽

Digital Format

AbstractThe isothermal molecular diagnosis with CRISPR has attracted particular interest for the sensitive, specific detection of nucleic acids. However, most of the assays with Cas enzymes were performed in bulk assays using multistep approaches and hard to realize quantitative detection. Herein, we report Microfluidics-Enabled Digital Isothermal Cas13a Assay (MEDICA), a digital format of SHERLOCK with enhanced robustness and sensitivity. We first address the macromolecular crowding problems when combining the recombinase polymerase amplification (RPA) and Cas13a detection into a one-pot SHERLOCK assay. After the assay optimization, the enhanced one-pot SHERLOCK (E-SHERLOCK) achieves high robustness and 200-fold increased sensitivity. Leveraging droplet microfluidics, we streamline the E-SHERLOCK to eliminate undesired input targets caused by pre-amplification before partition, enabling background-free absolute quantification. From the real-time monitoring, MEDICA enables qualitative detection within 10 min and absolute quantification within 25 min. For the proof of concept, we applied MEDICA to quantify HPV 16 and 18 viral loads in 44 clinical samples, indicating perfect accordance with qPCR results. MEDICA highlights the CRISPR-based isothermal assays are promising for the next generation of point-of-care diagnostics.

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Comparison of seven commercial SARS-CoV-2 rapid Point-of-Care Antigen tests

10.1101/2020.11.12.20230292 ◽

2020 ◽

Cited By ~ 9

Author(s):

Victor M. Corman ◽

Verena Claudia Haage ◽

Tobias Bleicker ◽

Marie Luisa Schmidt ◽

Barbara Mühlemann ◽

...

Keyword(s):

Limit Of Detection ◽

Point Of Care ◽

Rapid Test ◽

Infectious Period ◽

Clinical Samples ◽

Virus Concentration ◽

Viral Loads ◽

And Performance ◽

Antigen Tests ◽

Point Of Care Tests

AbstractBackgroundAntigen point of care tests (AgPOCT) can accelerate SARS-CoV-2 testing. As first AgPOCT are becoming available, there is a growing interest in their utility and performance.MethodsHere we compare AgPOCT products by seven suppliers: the Abbott Panbio™ COVID-19 Ag Rapid Test; the RapiGEN BIOCREDIT COVID-19 Ag; the Healgen® Coronavirus Ag Rapid Test Cassette (Swab); the Coris Bioconcept Covid.19 Ag Respi-Strip; the R-Biopharm RIDA®QUICK SARS-CoV-2 Antigen; the NAL von minden NADAL COVID19-Ag Test; and the Roche/SD Biosensor SARS-CoV Rapid Antigen Test. Tests were evaluated on recombinant nucleoprotein, cultured endemic and emerging coronaviruses, stored clinical samples with known SARS-CoV-2 viral loads (n=138), stored samples from patients with respiratory agents other than SARS-CoV-2 (n=100), as well as self-sampled swabs from healthy volunteers (n=35).FindingsLimits of detection in six of seven tested products ranged between 2.08 × 106 and 2.88 × 107 copies per swab, the outlier at 1.58 × 1010 copies per swab. Specificities ranged between 98.53% and 100% in five products, with two outliers at 94.85% and 88.24%. False positive results were not associated with any specific respiratory agent. As some of the tested AgPOCT were early production lots, the observed issues with specificity are unlikely to persist.InterpretationThe sensitivity range of most AgPOCT overlaps with viral load figures typically observed during the first week of symptoms, which marks the infectious period in the majority patients. AgPOCTs with a limit of detection that approximates the virus concentration above which patients are infectious may enable shortcuts in decision-making in various areas of healthcare and public health.

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Novel High Sensitivity Tuberculosis Point-of-Care Test for People Living with HIV

SSRN Electronic Journal ◽

10.2139/ssrn.3254479 ◽

2018 ◽

Cited By ~ 2

Author(s):

Tobias Broger ◽

Bianca Sossen ◽

Elloise du Toit ◽

Andrew D. Kerkhoff ◽

Charlotte Schutz ◽

...

Keyword(s):

Point Of Care ◽

High Sensitivity ◽

People Living With Hiv ◽

Point Of Care Test ◽

Living With Hiv

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Trends in Diagnosis for Active Tuberculosis Using Nanomaterials

Current Medicinal Chemistry ◽

10.2174/0929867325666180912105617 ◽

2019 ◽

Vol 26 (11) ◽

pp. 1946-1959 ◽

Cited By ~ 3

Author(s):

Le Minh Tu Phan ◽

Lemma Teshome Tufa ◽

Hwa-Jung Kim ◽

Jaebeom Lee ◽

Tae Jung Park

Keyword(s):

Sensitivity And Specificity ◽

High Efficiency ◽

Point Of Care ◽

High Sensitivity ◽

Signs And Symptoms ◽

Diagnostic Methods ◽

Advantages And Disadvantages ◽

Treatment And Prevention ◽

Clinical Tools ◽

Diagnostic Applications

Background:Tuberculosis (TB), one of the leading causes of death worldwide, is difficult to diagnose based only on signs and symptoms. Methods for TB detection are continuously being researched to design novel effective clinical tools for the diagnosis of TB.Objective:This article reviews the methods to diagnose TB at the latent and active stages and to recognize prospective TB diagnostic methods based on nanomaterials.Methods:The current methods for TB diagnosis were reviewed by evaluating their advantages and disadvantages. Furthermore, the trends in TB detection using nanomaterials were discussed regarding their performance capacity for clinical diagnostic applications.Results:Current methods such as microscopy, culture, and tuberculin skin test are still being employed to diagnose TB, however, a highly sensitive point of care tool without false results is still needed. The utilization of nanomaterials to detect the specific TB biomarkers with high sensitivity and specificity can provide a possible strategy to rapidly diagnose TB. Although it is challenging for nanodiagnostic platforms to be assessed in clinical trials, active TB diagnosis using nanomaterials is highly expected to achieve clinical significance for regular application. In addition, aspects and future directions in developing the high-efficiency tools to diagnose active TB using advanced nanomaterials are expounded.Conclusion:This review suggests that nanomaterials have high potential as rapid, costeffective tools to enhance the diagnostic sensitivity and specificity for the accurate diagnosis, treatment, and prevention of TB. Hence, portable nanobiosensors can be alternative effective tests to be exploited globally after clinical trial execution.

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