Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2
ABSTRACT At the end of 2019, a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused a pandemic that persists to date and has resulted in more than 6.2 million deaths. In the last couple of years, researchers have made great efforts to develop a diagnostic technique t...
| Main Authors: | , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
American Society for Microbiology
2022-10-01
|
| Series: | Microbiology Spectrum |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.02398-22 |
| _version_ | 1811329686717857792 |
|---|---|
| author | Concha Ortiz-Cartagena Laura Fernández-García Lucia Blasco Olga Pacios Inés Bleriot María López Rafael Cantón María Tomás |
| author_facet | Concha Ortiz-Cartagena Laura Fernández-García Lucia Blasco Olga Pacios Inés Bleriot María López Rafael Cantón María Tomás |
| author_sort | Concha Ortiz-Cartagena |
| collection | DOAJ |
| description | ABSTRACT At the end of 2019, a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused a pandemic that persists to date and has resulted in more than 6.2 million deaths. In the last couple of years, researchers have made great efforts to develop a diagnostic technique that maintains high levels of sensitivity and specificity, since an accurate and early diagnosis is required to minimize the prevalence of SARS-CoV-2 infection. In this context, CRISPR-Cas systems are proposed as promising tools for development as diagnostic techniques due to their high specificity, highlighting that Cas13 endonuclease discriminates single nucleotide changes and displays collateral activity against single-stranded RNA molecules. With the aim of improving the sensitivity of diagnosis, this technology is usually combined with isothermal preamplification reactions (SHERLOCK, DETECTR). Based on this, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP)-CRISPR-Cas13a method for SARS-CoV-2 virus detection in nasopharyngeal samples without using RNA extraction that exhibits 100% specificity and 83% sensitivity, as well as a positive predictive value (PPV) of 100% and negative predictive values (NPVs) of 100%, 81%, 79.1%, and 66.7% for cycle threshold (CT) values of <20, 20 to 30, >30 and overall, respectively. IMPORTANCE The coronavirus disease 2019 (COVID-19) crisis has driven the development of innovative molecular diagnosis methods, including CRISPR-Cas technology. In this work, we performed a protocol, working with RNA extraction kit-free samples and using RT-LAMP-CRISPR-Cas13a technology; our results place this method at the forefront of rapid and specific diagnostic methods for COVID-19 due to the high specificity (100%), sensitivity (83%), PPVs (100%), and NPVs (81% for high viral loads) obtained with clinical samples. |
| first_indexed | 2024-04-13T15:48:15Z |
| format | Article |
| id | doaj.art-0cf5f349c9ff4afba2ecf32d81d0e03c |
| institution | Directory Open Access Journal |
| issn | 2165-0497 |
| language | English |
| last_indexed | 2024-04-13T15:48:15Z |
| publishDate | 2022-10-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj.art-0cf5f349c9ff4afba2ecf32d81d0e03c2022-12-22T02:40:54ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972022-10-0110510.1128/spectrum.02398-22Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2Concha Ortiz-Cartagena0Laura Fernández-García1Lucia Blasco2Olga Pacios3Inés Bleriot4María López5Rafael Cantón6María Tomás7Translational and Multidisciplinary Microbiology (MicroTM), Biomedical Research Institute A Coruña (INIBIC), Microbiology Department, Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, SpainTranslational and Multidisciplinary Microbiology (MicroTM), Biomedical Research Institute A Coruña (INIBIC), Microbiology Department, Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, SpainTranslational and Multidisciplinary Microbiology (MicroTM), Biomedical Research Institute A Coruña (INIBIC), Microbiology Department, Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, SpainTranslational and Multidisciplinary Microbiology (MicroTM), Biomedical Research Institute A Coruña (INIBIC), Microbiology Department, Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, SpainTranslational and Multidisciplinary Microbiology (MicroTM), Biomedical Research Institute A Coruña (INIBIC), Microbiology Department, Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, SpainTranslational and Multidisciplinary Microbiology (MicroTM), Biomedical Research Institute A Coruña (INIBIC), Microbiology Department, Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, SpainSpanish Network for Research in Infectious Diseases (REIPI) and CIBER de Enfermedades Infecciosas (CIBERINFEC), Instituto de Salud Carlos III, Madrid, SpainTranslational and Multidisciplinary Microbiology (MicroTM), Biomedical Research Institute A Coruña (INIBIC), Microbiology Department, Hospital A Coruña (CHUAC), University of A Coruña (UDC), A Coruña, SpainABSTRACT At the end of 2019, a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused a pandemic that persists to date and has resulted in more than 6.2 million deaths. In the last couple of years, researchers have made great efforts to develop a diagnostic technique that maintains high levels of sensitivity and specificity, since an accurate and early diagnosis is required to minimize the prevalence of SARS-CoV-2 infection. In this context, CRISPR-Cas systems are proposed as promising tools for development as diagnostic techniques due to their high specificity, highlighting that Cas13 endonuclease discriminates single nucleotide changes and displays collateral activity against single-stranded RNA molecules. With the aim of improving the sensitivity of diagnosis, this technology is usually combined with isothermal preamplification reactions (SHERLOCK, DETECTR). Based on this, we developed a reverse transcription-loop-mediated isothermal amplification (RT-LAMP)-CRISPR-Cas13a method for SARS-CoV-2 virus detection in nasopharyngeal samples without using RNA extraction that exhibits 100% specificity and 83% sensitivity, as well as a positive predictive value (PPV) of 100% and negative predictive values (NPVs) of 100%, 81%, 79.1%, and 66.7% for cycle threshold (CT) values of <20, 20 to 30, >30 and overall, respectively. IMPORTANCE The coronavirus disease 2019 (COVID-19) crisis has driven the development of innovative molecular diagnosis methods, including CRISPR-Cas technology. In this work, we performed a protocol, working with RNA extraction kit-free samples and using RT-LAMP-CRISPR-Cas13a technology; our results place this method at the forefront of rapid and specific diagnostic methods for COVID-19 due to the high specificity (100%), sensitivity (83%), PPVs (100%), and NPVs (81% for high viral loads) obtained with clinical samples.https://journals.asm.org/doi/10.1128/spectrum.02398-22COVID-19SARS-CoV-2RT-LAMPCRISPR-Cas13CRISPR-Casdiagnosis |
| spellingShingle | Concha Ortiz-Cartagena Laura Fernández-García Lucia Blasco Olga Pacios Inés Bleriot María López Rafael Cantón María Tomás Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2 Microbiology Spectrum COVID-19 SARS-CoV-2 RT-LAMP CRISPR-Cas13 CRISPR-Cas diagnosis |
| title | Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2 |
| title_full | Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2 |
| title_fullStr | Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2 |
| title_full_unstemmed | Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2 |
| title_short | Reverse Transcription-Loop-Mediated Isothermal Amplification-CRISPR-Cas13a Technology as a Promising Diagnostic Tool for SARS-CoV-2 |
| title_sort | reverse transcription loop mediated isothermal amplification crispr cas13a technology as a promising diagnostic tool for sars cov 2 |
| topic | COVID-19 SARS-CoV-2 RT-LAMP CRISPR-Cas13 CRISPR-Cas diagnosis |
| url | https://journals.asm.org/doi/10.1128/spectrum.02398-22 |
| work_keys_str_mv | AT conchaortizcartagena reversetranscriptionloopmediatedisothermalamplificationcrisprcas13atechnologyasapromisingdiagnostictoolforsarscov2 AT laurafernandezgarcia reversetranscriptionloopmediatedisothermalamplificationcrisprcas13atechnologyasapromisingdiagnostictoolforsarscov2 AT luciablasco reversetranscriptionloopmediatedisothermalamplificationcrisprcas13atechnologyasapromisingdiagnostictoolforsarscov2 AT olgapacios reversetranscriptionloopmediatedisothermalamplificationcrisprcas13atechnologyasapromisingdiagnostictoolforsarscov2 AT inesbleriot reversetranscriptionloopmediatedisothermalamplificationcrisprcas13atechnologyasapromisingdiagnostictoolforsarscov2 AT marialopez reversetranscriptionloopmediatedisothermalamplificationcrisprcas13atechnologyasapromisingdiagnostictoolforsarscov2 AT rafaelcanton reversetranscriptionloopmediatedisothermalamplificationcrisprcas13atechnologyasapromisingdiagnostictoolforsarscov2 AT mariatomas reversetranscriptionloopmediatedisothermalamplificationcrisprcas13atechnologyasapromisingdiagnostictoolforsarscov2 |