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A Single-step Multiplex Quantitative Real Time Polymerase Chain Reaction Assay for Hepatitis C Virus Genotypes
Akanksha Singh1, Dhananjay Singh Mankotia1, Mohammad Irshad1
1Clinical Biochemistry Division, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi-110029, India.
Journal of Translational Internal Medicine
|July 7, 2017
Summary
A new multiplex quantitative real-time PCR (qPCR) assay offers a fast, specific, and sensitive method for determining hepatitis C virus (HCV) genotypes. This assay provides a reliable tool for routine laboratory use in guiding antiviral treatment strategies.
Area of Science:
- Virology
- Molecular Biology
- Clinical Diagnostics
Background:
- Hepatitis C virus (HCV) treatment efficacy varies by genotype, necessitating accurate genotyping for therapeutic planning.
- Existing HCV genotyping methods can be time-consuming or lack the required speed and reliability.
- A need exists for a rapid and dependable assay to determine HCV genotypes in patient sera.
Purpose of the Study:
- To develop a single-step multiplex quantitative real-time polymerase chain reaction (qPCR) assay for determining hepatitis C virus (HCV) genotypes.
- To establish a fast, specific, and sensitive method for HCV genotyping in clinical settings.
Main Methods:
- Design of primers and hydrolysis probes targeting conserved regions of the HCV genome (5' UTR, core, NS5b).
- Standardization and optimization of singleplex qPCR assays followed by development of a multiplex assay.
- Assessment of sensitivity, specificity, quantification cycle (Cq), and limit of detection (LOD) for both singleplex and multiplex assays.
- Comparison of the multiplex assay with sequence analysis and restriction fragment length polymorphism (RFLP).
- Application of the multiplex qPCR assay for genotyping in a panel of 98 HCV RNA-positive sera.
Main Results:
- The multiplex assay detected HCV genotypes 1 (26.53%), 3 (66.32%), and 4 (2.04%) in 98 patient sera.
- One sample showed mixed infection (genotype 1 and 3); five samples were undetectable.
- Genotypes 2, 5, and 6 were not detected in the tested sera.
- Results from the multiplex assay were comparable to singleplex qPCR and sequence analysis, demonstrating clear advantages over RFLP.
- Sensitivity, specificity, Cq, and LOD values were consistent between singleplex and multiplex assays.
Conclusions:
- The developed multiplex qPCR assay is a fast, specific, and sensitive method for HCV genotyping.
- This assay is suitable for routine laboratory implementation.
- It serves as a valuable tool for guiding hepatitis C antiviral treatment strategies.
