Establishment of simultaneous detection and quantitation of HCV-RNA by third generation intercalating dye real-time PCR
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 t...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Kulliyyah of Medicine, International Islamic University Malaysia
2018
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| Online Access: | http://irep.iium.edu.my/66273/ http://irep.iium.edu.my/66273/ http://irep.iium.edu.my/66273/1/MRS2018-32_Poster%20%20Third%20generation%20dye.pdf |
| Summary: | 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. |
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