scholarly journals Rapid, accurate, nucleobase detection using FnCas9

2020 ◽  
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.

scholarly journals Rapid, field-deployable nucleobase detection and identification using FnCas9

2020 ◽  
Author(s):  
Mohd. Azhar ◽  
Rhythm Phutela ◽  
Asgar Hussain Ansari ◽  
Dipanjali Sinha ◽  
Namrata Sharma ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Signal Detection ◽  
Point Of Care ◽  
Disease Outbreaks ◽  
Rapid Diagnosis ◽  
Clinical Prognosis ◽  
Infectious Disease Outbreaks ◽  
Dna And Rna ◽  
Detection And Identification ◽  
Detection Assay

Detection of pathogenic sequences or variants in DNA and RNA through a point-of-care diagnostic approach is valuable for rapid clinical prognosis. In recent times, CRISPR based detection of nucleic acids has provided an economical and quicker alternative to sequencing-based platforms which are often difficult to implement in the field. Here, we present FnCas9 Editor Linked Uniform Detection Assay (FELUDA) that employs a highly accurate enzymatic readout for detecting nucleotide sequences, identifying nucleobase identity and inferring zygosity with precision. We demonstrate that FELUDA output can be adapted to multiple signal detection platforms and can be quickly designed and deployed for versatile applications including rapid diagnosis during infectious disease outbreaks like COVID-19.

scholarly journals A Highly Automated Mobile Laboratory for On-site Molecular Diagnostics in the COVID-19 Pandemic

2021 ◽  
Vol 67 (4) ◽  
pp. 672-683
Author(s):  
Wanli Xing ◽  
Jiadao Wang ◽  
Chao Zhao ◽  
Han Wang ◽  
Liang Bai ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Nucleic Acid ◽  
Limit Of Detection ◽  
Disease Outbreaks ◽  
Clinical Samples ◽  
Acid Extraction ◽  
Robot Arm ◽  
Mobile Laboratory ◽  
Infectious Disease Outbreaks ◽  
Specimen Collection

Abstract Background Infectious disease outbreaks such as the COVID-19 (coronavirus disease 2019) pandemic call for rapid response and complete screening of the suspected community population to identify potential carriers of pathogens. Central laboratories rely on time-consuming sample collection methods that are rarely available in resource-limited settings. Methods We present a highly automated and fully integrated mobile laboratory for fast deployment in response to infectious disease outbreaks. The mobile laboratory was equipped with a 6-axis robot arm for automated oropharyngeal swab specimen collection; virus in the collected specimen was inactivated rapidly using an infrared heating module. Nucleic acid extraction and nested isothermal amplification were performed by a “sample in, answer out” laboratory-on-a-chip system, and the result was automatically reported by the onboard information platform. Each module was evaluated using pseudovirus or clinical samples. Results The mobile laboratory was stand-alone and self-sustaining and capable of on-site specimen collection, inactivation, analysis, and reporting. The automated sampling robot arm achieved sampling efficiency comparable to manual collection. The collected samples were inactivated in as short as 12 min with efficiency comparable to a water bath without damage to nucleic acid integrity. The limit of detection of the integrated microfluidic nucleic acid analyzer reached 150 copies/mL within 45 min. Clinical evaluation of the onboard microfluidic nucleic acid analyzer demonstrated good consistency with reverse transcription quantitative PCR with a κ coefficient of 0.979. Conclusions The mobile laboratory provides a promising solution for fast deployment of medical diagnostic resources at critical junctions of infectious disease outbreaks and facilitates local containment of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) transmission.

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

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.

scholarly journals Monitoring SARS-CoV-2 variants alterations in Nice neighborhoods by wastewater nanopore sequencing

2021 ◽  
Author(s):  
Geraldine Rios ◽  
Caroline Lacoux ◽  
Vianney Leclercq ◽  
Anna Diamant ◽  
Kevin Lebrigand ◽  
...  
Keyword(s):  
Treatment Plant ◽  
Low Frequency ◽  
Disease Outbreaks ◽  
Clinical Samples ◽  
Nanopore Sequencing ◽  
Infectious Disease Outbreaks ◽  
Epidemiological Tool ◽  
Frequency Comparison ◽  
Wastewater Samples ◽  
The City

Background: Wastewater surveillance has been proposed as an epidemiological tool to define the prevalence and evolution of the SARS-CoV-2 epidemics. However, most implemented SARS-CoV-2 wastewater surveillance projects were relying on qPCR measurement of virus titers and did not address the mutational spectrum of SARS-CoV-2 circulating in the population. Methods: We have implemented a nanopore RNA sequencing monitoring system in the city of Nice (France, 550,000 inhabitants). Between October 2020 and March 2021, we monthly analyzed the SARS-CoV-2 variants in 113 wastewater samples collected in the main wastewater treatment plant and 20 neighborhoods. Findings: We initially detected the lineages predominant in Europe at the end of 2020 (B.1.160, B.1.177, B.1.367, B.1.474, and B.1.221). In January, a localized emergence of a variant (Spike:A522S) of the B.1.1.7 lineage occurred in one neighborhood. It rapidly spread and became dominant all over the city. Other variants of concern (B.1.351, P.1) were also detected in some neighborhoods, but at low frequency. Comparison with individual clinical samples collected during the same week showed that wastewater sequencing correctly identified the same lineages as those found in COVID-19 patients. Interpretation: Wastewater sequencing allowed to document the diversity of SARS-CoV-2 sequences within the different neighborhoods of the city of Nice. Our results illustrate how sequencing of sewage samples can be used to track pathogen sequence diversity in the current pandemics and in future infectious disease outbreaks.

scholarly journals RNA viromics of Southern California wastewater and detection of SARS-CoV-2 single nucleotide variants.

2021 ◽  
Author(s):  
Jason A Rothman ◽  
Theresa B Loveless ◽  
Joseph Kapcia ◽  
Eric D Adams ◽  
Joshua A Steele ◽  
...  
Keyword(s):  
Public Health ◽  
Los Angeles ◽  
Southern California ◽  
Municipal Wastewater ◽  
Rna Viruses ◽  
Disease Outbreaks ◽  
Clinical Samples ◽  
Human Populations ◽  
Single Nucleotide Variants ◽  
Single Nucleotide

Abstract: Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early-warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the COVID-19 pandemic to track disease burden through analyses of SARS-CoV-2 RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the healthcare system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven treatment plants (WTPs), collected August 2020 - January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically-important SARS-CoV-2 variants, along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. Importance: Wastewater based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the COVID-19 pandemic to track the spread of SARS-CoV-2 without adding burden to healthcare systems. In this study, we used metatranscriptomics and RT-ddPCR to assay RNA viruses across Southern California wastewater from August 2020 - January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single nucleotide variants through sequencing. Likewise, WTPs harbored different viromes, and we detected other human pathogens such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.

scholarly journals RNA viromics of Southern California wastewater and detection of SARS-CoV-2 single nucleotide variants.

2021 ◽  
Author(s):  
Jason A. Rothman ◽  
Theresa B. Loveless ◽  
Joseph Kapcia ◽  
Eric D. Adams ◽  
Joshua A. Steele ◽  
...  
Keyword(s):  
Public Health ◽  
Los Angeles ◽  
Southern California ◽  
Municipal Wastewater ◽  
Rna Viruses ◽  
Disease Outbreaks ◽  
Clinical Samples ◽  
Human Populations ◽  
Single Nucleotide Variants ◽  
Single Nucleotide

Municipal wastewater provides an integrated sample of a diversity of human-associated microbes across a sewershed, including viruses. Wastewater-based epidemiology (WBE) is a promising strategy to detect pathogens and may serve as an early-warning system for disease outbreaks. Notably, WBE has garnered substantial interest during the COVID-19 pandemic to track disease burden through analyses of SARS-CoV-2 RNA. Throughout the COVID-19 outbreak, tracking SARS-CoV-2 in wastewater has been an important tool for understanding the spread of the virus. Unlike traditional sequencing of SARS-CoV-2 isolated from clinical samples, which adds testing burden to the healthcare system, in this study, metatranscriptomics was used to sequence virus directly from wastewater. Here, we present a study in which we explored RNA viral diversity through sequencing 94 wastewater influent samples across seven treatment plants (WTPs), collected August 2020 – January 2021, representing approximately 16 million people in Southern California. Enriched viral libraries identified a wide diversity of RNA viruses that differed between WTPs and over time, with detected viruses including coronaviruses, influenza A, and noroviruses. Furthermore, single nucleotide variants (SNVs) of SARS-CoV-2 were identified in wastewater and we measured proportions of overall virus and SNVs across several months. We detected several SNVs that are markers for clinically-important SARS-CoV-2 variants, along with SNVs of unknown function, prevalence, or epidemiological consequence. Our study shows the potential of WBE to detect viruses in wastewater and to track the diversity and spread of viral variants in urban and suburban locations, which may aid public health efforts to monitor disease outbreaks. Importance: Wastewater based epidemiology (WBE) can detect pathogens across sewersheds, which represents the collective waste of human populations. As there is a wide diversity of RNA viruses in wastewater, monitoring the presence of these viruses is useful for public health, industry, and ecological studies. Specific to public health, WBE has proven valuable during the COVID-19 pandemic to track the spread of SARS-CoV-2 without adding burden to healthcare systems. In this study, we used metatranscriptomics and RT-ddPCR to assay RNA viruses across Southern California wastewater from August 2020 – January 2021, representing approximately 16 million people from Los Angeles, Orange, and San Diego counties. We found that SARS-CoV-2 quantification in wastewater correlates well with county-wide COVID-19 case data, and that we can detect SARS-CoV-2 single nucleotide variants through sequencing. Likewise, WTPs harbored different viromes, and we detected other human pathogens such as noroviruses and adenoviruses, furthering our understanding of wastewater viral ecology.

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

2021 ◽  
Author(s):  
Beatrice Casati ◽  
Joseph Peter Verdi ◽  
Alexander Hempelmann ◽  
Maximilian Kittel ◽  
Andrea Gutierrez Klaebisch ◽  
...  
Keyword(s):  
Point Of Care ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Clinical Validation ◽  
High Demand ◽  
Critical Feature ◽  
Viral Loads ◽  
Reverse Transcription Quantitative Pcr ◽  
Second Year ◽  
Positive Population

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.

scholarly journals Microfluidics-Enabled Digital Isothermal Cas13a Assay

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.

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

2020 ◽  
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.

scholarly journals Infectious disease outbreaks in the African region: overview of events reported to the World Health Organization in 2018

2019 ◽  
Vol 147 ◽  
Author(s):  
F. Mboussou ◽  
P. Ndumbi ◽  
R. Ngom ◽  
Z. Kassamali ◽  
O. Ogundiran ◽  
...  
Keyword(s):  
Infectious Disease ◽  
Disease Onset ◽  
Disease Outbreaks ◽  
Case Fatality ◽  
World Health ◽  
African Region ◽  
Regional Office ◽  
Member States ◽  
Infectious Disease Outbreaks ◽  
The World

Abstract The WHO African region is characterised by the largest infectious disease burden in the world. We conducted a retrospective descriptive analysis using records of all infectious disease outbreaks formally reported to the WHO in 2018 by Member States of the African region. We analysed the spatio-temporal distribution, the notification delay as well as the morbidity and mortality associated with these outbreaks. In 2018, 96 new disease outbreaks were reported across 36 of the 47 Member States. The most commonly reported disease outbreak was cholera which accounted for 20.8% (n = 20) of all events, followed by measles (n = 11, 11.5%) and Yellow fever (n = 7, 7.3%). About a quarter of the outbreaks (n = 23) were reported following signals detected through media monitoring conducted at the WHO regional office for Africa. The median delay between the disease onset and WHO notification was 16 days (range: 0–184). A total of 107 167 people were directly affected including 1221 deaths (mean case fatality ratio (CFR): 1.14% (95% confidence interval (CI) 1.07%–1.20%)). The highest CFR was observed for diseases targeted for eradication or elimination: 3.45% (95% CI 0.89%–10.45%). The African region remains prone to outbreaks of infectious diseases. It is therefore critical that Member States improve their capacities to rapidly detect, report and respond to public health events.

Sign in / Sign up

Export Citation Format

Share Document