Establishment of simultaneous detection and quantitation of HCV-RNA by third generation intercalating dye real-time PCR

• Main Authors: M. Saleh Habil, Akrahm, Hamzah, Hairul Aini, Mustafa, Imad Al-Deen, A Talib, Noorlelawati, Abdul Rahman, Siti Nurul Fazlin
• Format: Article
• Language: English
• Published: Kulliyyah of Medicine, International Islamic University Malaysia 2018
• Subjects: QR Microbiology; QR355 Virology
• Online Access: http://irep.iium.edu.my/66273/1/MRS2018-32_Poster%20%20Third%20generation%20dye.pdf; http://irep.iium.edu.my/66273/; http://iiumedic.net/imjm/v1/download/vol_17_no_3_supp_1/MRS2018-32.pdf

Background: Rapid quantification of hepatitis C virus is helpful in determining and monitoring of the disease progression and nature of the virus replication. Objective: The aim of the present study was to establish a fast, specific and sensitive tool for HCV-RNA quantification. Methodology: 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 high analytical and diagnostic performance for HCV quantification, suggesting its potential in clinical diagnosis and management.