Multiplex real-time reverse transcription-PCR assay for determination of hepatitis C virus genotypes
A variety of methods have been used to determine hepatitis C virus (HCV) genotypes. Because therapeutic decisions for chronic HCV-related hepatitis are made on the basis of genotype, it is important that genotype be accurately determined by clinical laboratories. Existing methods are often subjective, inaccurate, manual, time-consuming, and contamination prone. We therefore evaluated real-time reverse transcription-PCR (RT-PCR) reagents that have recently become commercially available (Abbott HCV Genotype ASR). The assay developed by our laboratory starts with purified RNA and can be performed in 4 to 5 h. An initial evaluation of 479 samples was done with a restriction fragment length polymorphism (RFLP) method and the RT-PCR assay, and discrepant samples were sequenced. An additional 1,200 samples were then tested, and data from all assays were used to evaluate the efficiency and specificity of each genotype-specific reaction. Good correlation between results by the two methods was seen. Discrepant samples included those indeterminate by the RT-PCR assay (n = 110) and a subset that were incorrectly called 2a by the RFLP method (n = 75). The real-time RT-PCR assay performed well with genotype 1, 2, and 3 samples. Inadequate numbers of samples were available to evaluate fully genotypes 4, 5, and 6. Analysis of each primer-probe set demonstrated that weak cross-reactive amplifications were common but usually did not interfere with the genotype determination. However, in about 1% of samples, two or more genotypes amplified at roughly equivalent amounts. Further studies are necessary to determine whether these mixed-genotype samples are true mixtures or a reflection of occasional cross-reactive amplifications.
| Medienart: |
Artikel |
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| Erscheinungsjahr: |
2006 |
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| Erschienen: |
2006 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:44 |
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| Enthalten in: |
Journal of clinical microbiology - 44(2006), 11 vom: 03. Nov., Seite 4149-56 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Cook, Linda [VerfasserIn] |
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| Themen: |
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| Anmerkungen: |
Date Completed 22.12.2006 Date Revised 13.11.2018 published: Print-Electronic Citation Status MEDLINE |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM165482826 |
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| 245 | 1 | 0 | |a Multiplex real-time reverse transcription-PCR assay for determination of hepatitis C virus genotypes |
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| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a A variety of methods have been used to determine hepatitis C virus (HCV) genotypes. Because therapeutic decisions for chronic HCV-related hepatitis are made on the basis of genotype, it is important that genotype be accurately determined by clinical laboratories. Existing methods are often subjective, inaccurate, manual, time-consuming, and contamination prone. We therefore evaluated real-time reverse transcription-PCR (RT-PCR) reagents that have recently become commercially available (Abbott HCV Genotype ASR). The assay developed by our laboratory starts with purified RNA and can be performed in 4 to 5 h. An initial evaluation of 479 samples was done with a restriction fragment length polymorphism (RFLP) method and the RT-PCR assay, and discrepant samples were sequenced. An additional 1,200 samples were then tested, and data from all assays were used to evaluate the efficiency and specificity of each genotype-specific reaction. Good correlation between results by the two methods was seen. Discrepant samples included those indeterminate by the RT-PCR assay (n = 110) and a subset that were incorrectly called 2a by the RFLP method (n = 75). The real-time RT-PCR assay performed well with genotype 1, 2, and 3 samples. Inadequate numbers of samples were available to evaluate fully genotypes 4, 5, and 6. Analysis of each primer-probe set demonstrated that weak cross-reactive amplifications were common but usually did not interfere with the genotype determination. However, in about 1% of samples, two or more genotypes amplified at roughly equivalent amounts. Further studies are necessary to determine whether these mixed-genotype samples are true mixtures or a reflection of occasional cross-reactive amplifications | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 700 | 1 | |a Sullivan, KaWing |e verfasserin |4 aut | |
| 700 | 1 | |a Krantz, Elizabeth M |e verfasserin |4 aut | |
| 700 | 1 | |a Bagabag, Arthur |e verfasserin |4 aut | |
| 700 | 1 | |a Jerome, Keith R |e verfasserin |4 aut | |
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