Ultra-sensitive and high-throughput CRISPR-p owered COVID-19 diagnosis
Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-...
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| Published in: | Biosensors & bioelectronics Vol. 164; p. 112316 |
|---|---|
| Main Authors: | , , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
England
Elsevier B.V
15.09.2020
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| Subjects: | |
| ISSN: | 0956-5663, 1873-4235, 1873-4235 |
| Online Access: | Get full text |
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| Abstract | Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.
•Integrated CRISPR for COVID-19 clinical diagnosis.•Rapidly identify SARS-CoV-2-specific RNA signatures at an ultra-low concentration.•Detection environment friendly and compatible for high throughput COVID-19 screening in hospital.•Successful development and validation with swab samples from patients.•Improve diagnosis of individuals with suspected COVID-19 infections. |
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| AbstractList | Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.
•
Integrated CRISPR for COVID-19 clinical diagnosis.
•
Rapidly identify SARS-CoV-2-specific RNA signatures at an ultra-low concentration.
•
Detection environment friendly and compatible for high throughput COVID-19 screening in hospital.
•
Successful development and validation with swab samples from patients.
•
Improve diagnosis of individuals with suspected COVID-19 infections. Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts. Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts.Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts. Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts. •Integrated CRISPR for COVID-19 clinical diagnosis.•Rapidly identify SARS-CoV-2-specific RNA signatures at an ultra-low concentration.•Detection environment friendly and compatible for high throughput COVID-19 screening in hospital.•Successful development and validation with swab samples from patients.•Improve diagnosis of individuals with suspected COVID-19 infections. Recent research suggests that SARS-CoV-2-infected individuals can be highly infectious while asymptomatic or pre-symptomatic, and that an infected person may infect 5.6 other individuals on average. This situation highlights the need for rapid, sensitive SARS-CoV-2 diagnostic assays capable of high-throughput operation that can preferably utilize existing equipment to facilitate broad, large-scale screening efforts. We have developed a CRISPR-based assay that can meet all these criteria. This assay utilizes a custom CRISPR Cas12a/gRNA complex and a fluorescent probe to detect target amplicons produced by standard RT-PCR or isothermal recombinase polymerase amplification (RPA), to allow sensitive detection at sites not equipped with real-time PCR systems required for qPCR diagnostics. We found this approach allowed sensitive and robust detection of SARS-CoV-2 positive samples, with a sample-to-answer time of ~50 min, and a limit of detection of 2 copies per sample. CRISPR assay diagnostic results obtained nasal swab samples of individuals with suspected COVID-19 cases were comparable to paired results from a CDC-approved quantitative RT-PCR (RT-qPCR) assay performed in a state testing lab, and superior to those produced by same assay in a clinical lab, where the RT-qPCR assay exhibited multiple invalid or inconclusive results. Our assay also demonstrated greater analytical sensitivity and more robust diagnostic performance than other recently reported CRISPR-based assays. Based on these findings, we believe that a CRISPR-based fluorescent application has potential to improve current COVID-19 screening efforts. |
| ArticleNumber | 112316 |
| Author | Lin, Zhen Yin, Xiaoming Tian, Di Lyon, Christopher J. Drouin, Arnaud Fusco, Dahlene Liu, Yang Lai, Weihua Hu, Tony Ning, Bo Huang, Zhen |
| Author_xml | – sequence: 1 givenname: Zhen surname: Huang fullname: Huang, Zhen organization: Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA, 70112, USA – sequence: 2 givenname: Di surname: Tian fullname: Tian, Di organization: Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA, 70112, USA – sequence: 3 givenname: Yang surname: Liu fullname: Liu, Yang organization: Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA, 70112, USA – sequence: 4 givenname: Zhen surname: Lin fullname: Lin, Zhen organization: Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA, 70112, USA – sequence: 5 givenname: Christopher J. surname: Lyon fullname: Lyon, Christopher J. organization: Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA, 70112, USA – sequence: 6 givenname: Weihua surname: Lai fullname: Lai, Weihua organization: State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjin Road, Nanchang, 330047, China – sequence: 7 givenname: Dahlene surname: Fusco fullname: Fusco, Dahlene organization: Departments of Medicine and Pathology, Tulane University School of Medicine, 333 S Liberty St New Orleans, LA, 70114, USA – sequence: 8 givenname: Arnaud surname: Drouin fullname: Drouin, Arnaud organization: Departments of Medicine and Pathology, Tulane University School of Medicine, 333 S Liberty St New Orleans, LA, 70114, USA – sequence: 9 givenname: Xiaoming surname: Yin fullname: Yin, Xiaoming organization: Department of Pathology and Laboratory Medicine, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA, 70112, USA – sequence: 10 givenname: Tony surname: Hu fullname: Hu, Tony email: tonyhu@tulane.edu organization: Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA, 70112, USA – sequence: 11 givenname: Bo surname: Ning fullname: Ning, Bo email: bning1@tulane.edu organization: Center for Cellular and Molecular Diagnostics, Tulane University School of Medicine, 1430 Tulane Ave., New Orleans, LA, 70112, USA |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/32553350$$D View this record in MEDLINE/PubMed |
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| SubjectTerms | Base Sequence Betacoronavirus - genetics Betacoronavirus - isolation & purification Biosensing Techniques - methods Biosensing Techniques - statistics & numerical data biosensors Coronavirus infections Coronavirus Infections - diagnosis Coronavirus Infections - virology COVID-19 CRISPR CRISPR-Cas Systems detection limit diagnostic techniques fluorescence Fluorescent detection Fluorescent Dyes gene editing Genes, Viral High-Throughput Nucleotide Sequencing - methods High-Throughput Nucleotide Sequencing - statistics & numerical data Highly sensitive Humans Molecular diagnosis nose Nucleic Acid Amplification Techniques - methods Nucleic Acid Amplification Techniques - statistics & numerical data Pandemics Pneumonia, Viral - diagnosis Pneumonia, Viral - virology Predictive Value of Tests quantitative polymerase chain reaction recombinases Reproducibility of Results reverse transcriptase polymerase chain reaction Reverse Transcriptase Polymerase Chain Reaction - methods Reverse Transcriptase Polymerase Chain Reaction - statistics & numerical data RNA, Viral - analysis RNA, Viral - genetics SARS-CoV-2 screening Sensitivity and Specificity |
| Title | Ultra-sensitive and high-throughput CRISPR-p owered COVID-19 diagnosis |
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