Multiplexing of reverse-transcription polymerase chain reaction for Hepatitis C virus genotyping
Background: Hepatitis C genotyping is a mandatory for the treatment and management of patients who are advised for interferon therapy. The gold standard of HCV genotyping is based on reverse-transcription polymerase chain reaction (RT-PCR), followed by DNA sequencing. Objective: We improved our tw...
| Main Authors: | , , |
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| Format: | Proceeding Paper |
| Language: | English |
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2013
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| Online Access: | http://irep.iium.edu.my/35014/ http://irep.iium.edu.my/35014/2/USIM_Annual_Health_Conference.pdf |
| _version_ | 1848780996852318208 |
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| author | Hamzah, Hairul Aini Mustafa Mahmoud, Mohammed Imad Al-Deen Talib, Norlelawati A. |
| author_facet | Hamzah, Hairul Aini Mustafa Mahmoud, Mohammed Imad Al-Deen Talib, Norlelawati A. |
| author_sort | Hamzah, Hairul Aini |
| building | IIUM Repository |
| collection | Online Access |
| description | Background: Hepatitis C genotyping is a mandatory for the treatment and management of patients who are advised for interferon therapy. The gold standard of HCV genotyping is based on reverse-transcription polymerase chain reaction (RT-PCR), followed by DNA sequencing.
Objective: We improved our two-tube format RT-PCR to one-tube for the HCV cDNA amplification. We also optimized the RT-PCR reaction for multiplexing purpose.
Methodology: Two-tube assay was evaluated using a positive control with known HCV viral load (2,148,000 IU/ml), which was determined at Gribbles Laboratory (Australia). Optimization of one-tube format and multiplex RT-PCR were carried out using Superscript III One-Step RT-PCR System (Invitrogen, USA), targeting the universal 5’UTR and NS5B regions.
Results: One-tube format was easily carried out using Superscript III One-Step RT-PCR System (Invitrogen, USA). However, multiplex system was not feasible with the optimized one-tube format. Multiplex RT-PCR was successfully performed with the undiluted HCV-RNA. The bands (414 bp & 212 bp) could be cut simultaneously for the gel extraction and purification using MinElute Gel purification System (Qiagen, Germany) prior to cDNA sequencing. HCV genotype 3, 1 and 4 could be determined by the assay but not HCV genotype 6.
Conclusion: Since the majority of HCV strains in Malaysia are derived from the HCV genotype 3 and 1, this method can be used for rapid genotyping purposes.
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| first_indexed | 2025-11-14T15:42:33Z |
| format | Proceeding Paper |
| id | iium-35014 |
| institution | International Islamic University Malaysia |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T15:42:33Z |
| publishDate | 2013 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | iium-350142014-01-28T04:58:10Z http://irep.iium.edu.my/35014/ Multiplexing of reverse-transcription polymerase chain reaction for Hepatitis C virus genotyping Hamzah, Hairul Aini Mustafa Mahmoud, Mohammed Imad Al-Deen Talib, Norlelawati A. QR355 Virology Background: Hepatitis C genotyping is a mandatory for the treatment and management of patients who are advised for interferon therapy. The gold standard of HCV genotyping is based on reverse-transcription polymerase chain reaction (RT-PCR), followed by DNA sequencing. Objective: We improved our two-tube format RT-PCR to one-tube for the HCV cDNA amplification. We also optimized the RT-PCR reaction for multiplexing purpose. Methodology: Two-tube assay was evaluated using a positive control with known HCV viral load (2,148,000 IU/ml), which was determined at Gribbles Laboratory (Australia). Optimization of one-tube format and multiplex RT-PCR were carried out using Superscript III One-Step RT-PCR System (Invitrogen, USA), targeting the universal 5’UTR and NS5B regions. Results: One-tube format was easily carried out using Superscript III One-Step RT-PCR System (Invitrogen, USA). However, multiplex system was not feasible with the optimized one-tube format. Multiplex RT-PCR was successfully performed with the undiluted HCV-RNA. The bands (414 bp & 212 bp) could be cut simultaneously for the gel extraction and purification using MinElute Gel purification System (Qiagen, Germany) prior to cDNA sequencing. HCV genotype 3, 1 and 4 could be determined by the assay but not HCV genotype 6. Conclusion: Since the majority of HCV strains in Malaysia are derived from the HCV genotype 3 and 1, this method can be used for rapid genotyping purposes. 2013 Proceeding Paper PeerReviewed application/pdf en http://irep.iium.edu.my/35014/2/USIM_Annual_Health_Conference.pdf Hamzah, Hairul Aini and Mustafa Mahmoud, Mohammed Imad Al-Deen and Talib, Norlelawati A. (2013) Multiplexing of reverse-transcription polymerase chain reaction for Hepatitis C virus genotyping. In: USIM's 4th Annual Health Conference (AHC 2013), 5 - 6 October 2013, Hotel Istana, Kuala Lumpur. |
| spellingShingle | QR355 Virology Hamzah, Hairul Aini Mustafa Mahmoud, Mohammed Imad Al-Deen Talib, Norlelawati A. Multiplexing of reverse-transcription polymerase chain reaction for Hepatitis C virus genotyping |
| title | Multiplexing of reverse-transcription polymerase chain reaction for Hepatitis C virus genotyping |
| title_full | Multiplexing of reverse-transcription polymerase chain reaction for Hepatitis C virus genotyping |
| title_fullStr | Multiplexing of reverse-transcription polymerase chain reaction for Hepatitis C virus genotyping |
| title_full_unstemmed | Multiplexing of reverse-transcription polymerase chain reaction for Hepatitis C virus genotyping |
| title_short | Multiplexing of reverse-transcription polymerase chain reaction for Hepatitis C virus genotyping |
| title_sort | multiplexing of reverse-transcription polymerase chain reaction for hepatitis c virus genotyping |
| topic | QR355 Virology |
| url | http://irep.iium.edu.my/35014/ http://irep.iium.edu.my/35014/2/USIM_Annual_Health_Conference.pdf |