Development of simple, rapid, and sensitive methods for detection of hepatitis C virus RNA from whole blood using reverse transcription loop-mediated isothermal amplification
Hepatitis C virus (HCV) infection is an underdiagnosed global health problem. Diagnosis of current HCV infections typically requires testing for HCV RNA using high-complexity laboratory tests. Methods for the detection of HCV RNA that are simple, inexpensive, rapid, and compatible with use outside of a laboratory setting are very important in order to improve access to hepatitis C diagnostic testing and facilitate accelerated linkage to care. We developed and evaluated three simple workflows for extracting HCV RNA from small volumes of whole blood for use in a sensitive, pan-genotypic RT-LAMP assay. The water workflow uses osmotic stress to release HCV RNA and has a limit of detection of 4.3 log10(IU/mL) (95% CI 4.0-4.9). The heat workflow uses a heating step to release HCV RNA and has a limit of detection of 4.2 log10(IU/mL) (95% CI 3.8-5.1). The bead workflow, which uses chemical lysis of the sample and a streamlined paramagnetic solid phase reversible immobilization bead procedure for nucleic acid purification, has a limit of detection of 2.8 log10(IU/mL) (95% CI 2.5-3.4). When used to test whole blood spiked with HCV RNA-positive plasma samples in which most HCV levels were below 5.0 log10(IU/mL), the water, heat, and bead workflows detected HCV RNA in 69%, 75%, and 94% of samples, respectively. These workflows are compatible with visual lateral flow dipsticks, and each takes less than 60 min from sample to result. Each workflow can be performed with minimal and inexpensive equipment. With further procedural simplifications, these workflows may form the basis of assays for the point-of-care diagnosis of HCV infections.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:61 |
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| Enthalten in: |
Journal of clinical microbiology - 61(2023), 11 vom: 21. Nov., Seite e0077123 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Pauly, Matthew D [VerfasserIn] |
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| Links: |
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| Themen: |
059QF0KO0R |
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| Anmerkungen: |
Date Completed 27.11.2023 Date Revised 02.02.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1128/jcm.00771-23 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM364257997 |
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| 100 | 1 | |a Pauly, Matthew D |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Development of simple, rapid, and sensitive methods for detection of hepatitis C virus RNA from whole blood using reverse transcription loop-mediated isothermal amplification |
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| 500 | |a Date Completed 27.11.2023 | ||
| 500 | |a Date Revised 02.02.2024 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Hepatitis C virus (HCV) infection is an underdiagnosed global health problem. Diagnosis of current HCV infections typically requires testing for HCV RNA using high-complexity laboratory tests. Methods for the detection of HCV RNA that are simple, inexpensive, rapid, and compatible with use outside of a laboratory setting are very important in order to improve access to hepatitis C diagnostic testing and facilitate accelerated linkage to care. We developed and evaluated three simple workflows for extracting HCV RNA from small volumes of whole blood for use in a sensitive, pan-genotypic RT-LAMP assay. The water workflow uses osmotic stress to release HCV RNA and has a limit of detection of 4.3 log10(IU/mL) (95% CI 4.0-4.9). The heat workflow uses a heating step to release HCV RNA and has a limit of detection of 4.2 log10(IU/mL) (95% CI 3.8-5.1). The bead workflow, which uses chemical lysis of the sample and a streamlined paramagnetic solid phase reversible immobilization bead procedure for nucleic acid purification, has a limit of detection of 2.8 log10(IU/mL) (95% CI 2.5-3.4). When used to test whole blood spiked with HCV RNA-positive plasma samples in which most HCV levels were below 5.0 log10(IU/mL), the water, heat, and bead workflows detected HCV RNA in 69%, 75%, and 94% of samples, respectively. These workflows are compatible with visual lateral flow dipsticks, and each takes less than 60 min from sample to result. Each workflow can be performed with minimal and inexpensive equipment. With further procedural simplifications, these workflows may form the basis of assays for the point-of-care diagnosis of HCV infections | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, U.S. Gov't, Non-P.H.S. | |
| 650 | 4 | |a RNA detection | |
| 650 | 4 | |a assay development | |
| 650 | 4 | |a hepatitis C virus | |
| 650 | 4 | |a point of care | |
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| 700 | 1 | |a Hayden, Tonya M |e verfasserin |4 aut | |
| 700 | 1 | |a Ganova-Raeva, Lilia M |e verfasserin |4 aut | |
| 700 | 1 | |a Kamili, Saleem |e verfasserin |4 aut | |
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