Open AccessEstablishment of Simultaneous Detection and Quantitation of HCVRNA by Third Generation Intercalating Dye Real-time PCR
Author(s) -
Akrahm M. Saleh Habil,
Hairul Aini Hamzah,
Muhammad Imad Al-Deen Mustafa,
Norlelawati A. Talib,
Siti Nurul Fazlin Abdul Rahman
Publication year - 2018
Publication title -
international medical journal malaysia/iium medical journal malaysia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.139
H-Index - 9
eISSN - 2735-2285
pISSN - 1823-4631
DOI - 10.31436/imjm.v17i1.843
Subject(s) - medicine , real time polymerase chain reaction , detection limit , hepatitis c virus , chromatography , virology , virus , microbiology and biotechnology , biology , gene , genetics , chemistry
Rapid quantification of hepatitis C virus is helpful in determining and monitoring of the disease progression and nature of the virus replication. The aim of the present study was to establish a fast, specific and sensitive tool for HCVRNA quantification. Materials and Methods: A total of 50 serum samples, comprising of 40 HCV-positive and 10 HCV-negative, were included in our study. RNA was extracted, reverse transcribed, and then subjected to real-time PCR amplification. Real-time PCR using EvaGreen dye and primers targeting a 5’UTR was carried out. 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 developed real time PCR using EvaGreen dye has demonstrated a highly analytical and diagnostic performance for HCV quantification, suggesting its potential in clinical diagnosis and management.