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Multiplexing of reverse-transcription polymerase chain reaction for Hepatitis C virus genotyping

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.

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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.

Item Type: Conference or Workshop Item (Poster)
Additional Information: 5342/35014
Subjects: Q Science > QR Microbiology > QR355 Virology
Kulliyyahs/Centres/Divisions/Institutes (Can select more than one option. Press CONTROL button): Kulliyyah of Medicine > Department of Basic Medical
Depositing User: Dr Hairul Aini Hamzah
Date Deposited: 28 Jan 2014 12:58
Last Modified: 28 Jan 2014 12:58
URI: http://irep.iium.edu.my/id/eprint/35014

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