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Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay

Hepatitis C virus (HCV) infection can be cured with pan-genotypic direct-acting antiviral agents. However, identifying individuals with current hepatitis C remains a major challenge, especially in resource-limited settings where access to or availability of molecular tests is still limited. The goal...

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Autores principales: Kham-Kjing, Nang, Ngo-Giang-Huong, Nicole, Tragoolpua, Khajornsak, Khamduang, Woottichai, Hongjaisee, Sayamon
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9317538/
https://www.ncbi.nlm.nih.gov/pubmed/35885430
http://dx.doi.org/10.3390/diagnostics12071524
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author Kham-Kjing, Nang
Ngo-Giang-Huong, Nicole
Tragoolpua, Khajornsak
Khamduang, Woottichai
Hongjaisee, Sayamon
author_facet Kham-Kjing, Nang
Ngo-Giang-Huong, Nicole
Tragoolpua, Khajornsak
Khamduang, Woottichai
Hongjaisee, Sayamon
author_sort Kham-Kjing, Nang
collection PubMed
description Hepatitis C virus (HCV) infection can be cured with pan-genotypic direct-acting antiviral agents. However, identifying individuals with current hepatitis C remains a major challenge, especially in resource-limited settings where access to or availability of molecular tests is still limited. The goal of this study was to develop and validate a molecular assay for the rapid detection of HCV RNA in resource-limited settings. It is based on a combination of reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 12a (CRISPR–Cas12a) cleavage assay that allows the recognition of specific HCV nucleic acid sequences. Amplified products after the cleavage reactions can be visualized on lateral flow strips or measured with a fluorescence detector. When tested on clinical samples from individuals infected with HCV, HIV, or HBV, or from healthy donors, the RT-LAMP-coupled CRISPR–Cas12 assay yielded 96% sensitivity, 100% specificity, and 97% agreement as compared to the reference method (Roche COBAS AmpliPrep/COBAS TaqMan HCV Test). This assay could detect HCV RNA concentrations as low as 10 ng/µL (an estimated 2.38 Log(10) IU/mL). Therefore, this sensitive and specific assay may represent an affordable and reliable point-of-care test for the identification of individuals with active hepatitis C in low-resource settings.
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spelling pubmed-93175382022-07-27 Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay Kham-Kjing, Nang Ngo-Giang-Huong, Nicole Tragoolpua, Khajornsak Khamduang, Woottichai Hongjaisee, Sayamon Diagnostics (Basel) Article Hepatitis C virus (HCV) infection can be cured with pan-genotypic direct-acting antiviral agents. However, identifying individuals with current hepatitis C remains a major challenge, especially in resource-limited settings where access to or availability of molecular tests is still limited. The goal of this study was to develop and validate a molecular assay for the rapid detection of HCV RNA in resource-limited settings. It is based on a combination of reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 12a (CRISPR–Cas12a) cleavage assay that allows the recognition of specific HCV nucleic acid sequences. Amplified products after the cleavage reactions can be visualized on lateral flow strips or measured with a fluorescence detector. When tested on clinical samples from individuals infected with HCV, HIV, or HBV, or from healthy donors, the RT-LAMP-coupled CRISPR–Cas12 assay yielded 96% sensitivity, 100% specificity, and 97% agreement as compared to the reference method (Roche COBAS AmpliPrep/COBAS TaqMan HCV Test). This assay could detect HCV RNA concentrations as low as 10 ng/µL (an estimated 2.38 Log(10) IU/mL). Therefore, this sensitive and specific assay may represent an affordable and reliable point-of-care test for the identification of individuals with active hepatitis C in low-resource settings. MDPI 2022-06-23 /pmc/articles/PMC9317538/ /pubmed/35885430 http://dx.doi.org/10.3390/diagnostics12071524 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Kham-Kjing, Nang
Ngo-Giang-Huong, Nicole
Tragoolpua, Khajornsak
Khamduang, Woottichai
Hongjaisee, Sayamon
Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay
title Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay
title_full Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay
title_fullStr Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay
title_full_unstemmed Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay
title_short Highly Specific and Rapid Detection of Hepatitis C Virus Using RT-LAMP-Coupled CRISPR–Cas12 Assay
title_sort highly specific and rapid detection of hepatitis c virus using rt-lamp-coupled crispr–cas12 assay
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9317538/
https://www.ncbi.nlm.nih.gov/pubmed/35885430
http://dx.doi.org/10.3390/diagnostics12071524
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