Hepatitis c virus RNA quantification using Evagreen® dye real time PCR

Background: HCV exhibits extreme genome heterogeneity, additionally it usually present in blood at a low copy number. Thus, high specificity and sensitivity represent a major challenge during development of any HCV detection and quantification assay.. Objective: The aim of the present study was to...

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Main Authors:	Hamzah, Hairul Aini, M. Saleh Habil, Akrahm, Mustafa Mahmoud, Mohammed Imad Al-Deen, A.Talib, Norlelawati, Hasmoni, Mohamed Hadzri
Format:	Conference or Workshop Item
Language:	English
Published:	2016
Subjects:	QR Microbiology R Medicine (General)
Online Access:	http://irep.iium.edu.my/63758/ http://irep.iium.edu.my/63758/1/63758_HEPATITIS%20C%20VIRUS%20RNA.pdf

id	iium-63758
recordtype	eprints
spelling	iium-637582018-06-29T03:28:24Z http://irep.iium.edu.my/63758/ Hepatitis c virus RNA quantification using Evagreen® dye real time PCR Hamzah, Hairul Aini M. Saleh Habil, Akrahm Mustafa Mahmoud, Mohammed Imad Al-Deen A.Talib, Norlelawati Hasmoni, Mohamed Hadzri QR Microbiology R Medicine (General) Background: HCV exhibits extreme genome heterogeneity, additionally it usually present in blood at a low copy number. Thus, high specificity and sensitivity represent a major challenge during development of any HCV detection and quantification assay.. Objective: The aim of the present study was to establish a highly specific and sensitive semi-nested real time PCR using EvaGreen dye for detecting and quantifying HCV RNA. Methodology: This is a laboratory cross-sectional study. A total of 50 serum samples, comprising 40 HCV- positive and 10 HCV-negative, were included in our study. RNA was extracted, reverse transcribed, and then subjected to two rounds of PCR amplification. In the first round, conventional PCR was performed using a pair of primers targeting the 5'UTR. In the second round, real time PCR using EvaGreen dye and primers targeting a region within the previously amplified product was carried out for sensitive detection and quantification. Reference samples with known viral load were treated similarly to the unknown samples and used to create the standard curves. Results: Our method showed a high level of analytical specificity and accuracy, with a low limit of detection (~2 IU/ml). It yielded repeatable results with less than 4% of intra- assay variation. The assay covered a broad dynamic range of quantification, ranging from 0.34 to 6 log IU/ml. The diagnostic sensitivity, specificity, and accuracy were all 100%, indicating neither false positive nor false negative results were obtained. Conclusion: The newly developed semi-nested real time PCR using EvaGreen dye has demonstrated a high analytical and diagnostic performance for HCV viral load, suggesting its potential to have wide applications in clinical diagnosis and therapeutic management. Key words: Hepatitis C, Real-time PCR, viral load 2016-12-07 Conference or Workshop Item PeerReviewed application/pdf en http://irep.iium.edu.my/63758/1/63758_HEPATITIS%20C%20VIRUS%20RNA.pdf Hamzah, Hairul Aini and M. Saleh Habil, Akrahm and Mustafa Mahmoud, Mohammed Imad Al-Deen and A.Talib, Norlelawati and Hasmoni, Mohamed Hadzri (2016) Hepatitis c virus RNA quantification using Evagreen® dye real time PCR. In: Medical, Medicine & Health Sciences MMHS 2016, 7th-8th Dec.2016, Bali, Indonesia.
repository_type	Digital Repository
institution_category	Local University
institution	International Islamic University Malaysia
building	IIUM Repository
collection	Online Access
language	English
topic	QR Microbiology R Medicine (General)
spellingShingle	QR Microbiology R Medicine (General) Hamzah, Hairul Aini M. Saleh Habil, Akrahm Mustafa Mahmoud, Mohammed Imad Al-Deen A.Talib, Norlelawati Hasmoni, Mohamed Hadzri Hepatitis c virus RNA quantification using Evagreen® dye real time PCR
description	Background: HCV exhibits extreme genome heterogeneity, additionally it usually present in blood at a low copy number. Thus, high specificity and sensitivity represent a major challenge during development of any HCV detection and quantification assay.. Objective: The aim of the present study was to establish a highly specific and sensitive semi-nested real time PCR using EvaGreen dye for detecting and quantifying HCV RNA. Methodology: This is a laboratory cross-sectional study. A total of 50 serum samples, comprising 40 HCV- positive and 10 HCV-negative, were included in our study. RNA was extracted, reverse transcribed, and then subjected to two rounds of PCR amplification. In the first round, conventional PCR was performed using a pair of primers targeting the 5'UTR. In the second round, real time PCR using EvaGreen dye and primers targeting a region within the previously amplified product was carried out for sensitive detection and quantification. Reference samples with known viral load were treated similarly to the unknown samples and used to create the standard curves. Results: Our method showed a high level of analytical specificity and accuracy, with a low limit of detection (~2 IU/ml). It yielded repeatable results with less than 4% of intra- assay variation. The assay covered a broad dynamic range of quantification, ranging from 0.34 to 6 log IU/ml. The diagnostic sensitivity, specificity, and accuracy were all 100%, indicating neither false positive nor false negative results were obtained. Conclusion: The newly developed semi-nested real time PCR using EvaGreen dye has demonstrated a high analytical and diagnostic performance for HCV viral load, suggesting its potential to have wide applications in clinical diagnosis and therapeutic management. Key words: Hepatitis C, Real-time PCR, viral load
format	Conference or Workshop Item
author	Hamzah, Hairul Aini M. Saleh Habil, Akrahm Mustafa Mahmoud, Mohammed Imad Al-Deen A.Talib, Norlelawati Hasmoni, Mohamed Hadzri
author_facet	Hamzah, Hairul Aini M. Saleh Habil, Akrahm Mustafa Mahmoud, Mohammed Imad Al-Deen A.Talib, Norlelawati Hasmoni, Mohamed Hadzri
author_sort	Hamzah, Hairul Aini
title	Hepatitis c virus RNA quantification using Evagreen® dye real time PCR
title_short	Hepatitis c virus RNA quantification using Evagreen® dye real time PCR
title_full	Hepatitis c virus RNA quantification using Evagreen® dye real time PCR
title_fullStr	Hepatitis c virus RNA quantification using Evagreen® dye real time PCR
title_full_unstemmed	Hepatitis c virus RNA quantification using Evagreen® dye real time PCR
title_sort	hepatitis c virus rna quantification using evagreen® dye real time pcr
publishDate	2016
url	http://irep.iium.edu.my/63758/ http://irep.iium.edu.my/63758/1/63758_HEPATITIS%20C%20VIRUS%20RNA.pdf
first_indexed	2023-09-18T21:30:25Z
last_indexed	2023-09-18T21:30:25Z
_version_	1777412473419202560

Hepatitis c virus RNA quantification using Evagreen® dye real time PCR

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