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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...

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Main Authors: Hamzah, Hairul Aini, Mustafa Mahmoud, Mohammed Imad Al-Deen, Talib, Norlelawati A.
Format: Conference or Workshop Item
Language:English
Published: 2013
Subjects:
QR355 Virology
Online Access:http://irep.iium.edu.my/35014/2/USIM_Annual_Health_Conference.pdf
http://irep.iium.edu.my/35014/
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spelling my.iium.irep.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 Conference or Workshop Item REM 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.
institution Universiti Islam Antarabangsa Malaysia
building IIUM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider International Islamic University Malaysia
content_source IIUM Repository (IREP)
url_provider http://irep.iium.edu.my/
language English
topic QR355 Virology
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
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.
format Conference or Workshop Item
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
title 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_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_sort multiplexing of reverse-transcription polymerase chain reaction for hepatitis c virus genotyping
publishDate 2013
url http://irep.iium.edu.my/35014/2/USIM_Annual_Health_Conference.pdf
http://irep.iium.edu.my/35014/
_version_ 1643610721520451584
score 13.211869

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