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Updated: Nov 12, 2025
Author Spotlight: Advancements and Challenges in Hepatitis B Virus Detection
Published on: December 15, 2023
Detection of circulating HCV recombinant form RF1_2k/1b in blood serum of patients by real-time RT-PCR
Ivan Alekseevich Akimov1, D I Timofeev1, A R Mavzyutov2,3
1AO «Vector-Best».
Insights
A new RT-PCR method accurately detects the hepatitis C virus (HCV) recombinant RF1_2k/1b, often misclassified by commercial kits. This improves treatment for millions affected by chronic hepatitis C complications.
Area of Science:
- Virology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Hepatitis C virus (HCV) infection affects 70 million globally, causing 400,000 deaths annually.
- Accurate HCV genotyping is crucial for effective antiviral treatment, as drug efficacy varies by viral genotype.
- Prevalent HCV recombinant form RF1_2k/1b is frequently misclassified as genotype 2 by commercial kits.
Purpose of the Study:
- To develop and validate a reliable method for detecting HCV RF1_2k/1b RNA in patient blood samples.
- To address the misclassification of RF1_2k/1b by existing commercial HCV genotyping kits.
- To improve the accuracy of HCV typing for appropriate patient management.
Main Methods:
- Developed a reverse transcription-polymerase chain reaction (RT-PCR) assay targeting two HCV genome regions (5'UTR and NS5b).
- Tested the method on 240 blood serum samples from HCV-infected patients previously genotyped by commercial kits.
- Validated results using Sanger sequencing and phylogenetic analysis.
Main Results:
- The new RT-PCR method identified 50 (20.8%) cases of RF1_2k/1b among samples classified as genotype 2 or mixed by commercial kits.
- Three mixed infections involving RF1_2k/1b and other subtypes (1a, 3a, 1b) were accurately identified.
- The method demonstrated high accuracy, confirmed by Sanger sequencing and phylogenetic analysis.
Conclusions:
- The developed RT-PCR method accurately identifies the prevalent HCV recombinant RF1_2k/1b.
- This method can overcome the limitations of commercial kits in correctly classifying HCV genotypes.
- Implementation in clinical settings can lead to more precise diagnosis and optimized treatment strategies for hepatitis C patients.
Abstract:
Globally, about 70 million people are infected with the hepatitis C virus (HCV), and about 400 thousand people die annually from chronic hepatitis C complications. The management of patients with chronic hepatitis C may require HCV genotyping, since the efficiency of some widely used antiviral drugs strongly depend on the viral genotype and/or subtype. The most prevalent HCV circulating recombinant form, RF1_2k/1b, is misclassified as genotype 2 by many commercial HCV genotyping kits, based on the RT-PCR analysis of the 5' untranslated region of the HCV genome. This leads to inappropriate patient treatment, since the accepted treatment schemes for HCV genotype 2 are ineffective for the RF1_2k/1b. Here we describe a method for detecting the RNA HCV RF1_2k/1b in blood samples by RT-PCR analysis of two regions in HCV genome (5'UTR and NS5b). The method was tested on 240 blood serum samples from HCV infected patients, in which HCV genotype was defined as 2 or mixed (2+1 or 2+3) by the two commercial genotyping kits "OT-Hepatogen-C genotype" ("DNA-Technology", Moscow) and "RealBest RNA HCV-1/2/3" ("Vector- Best ", Novosibirsk). 50 (20.8%) RF1_2k/1b cases were revealed, including three mixed infections: RF1_2k/1b + 1a, RF1_2k/1b + 3a, RF1_2k/1b + 1b. In all cases, the accuracy of HCV typing by the proposed method was confirmed by Sanger sequencing and phylogenetic analysis. The method is easy to implement into clinical practice and may be used in clinical settings equipped for RT-PCR analysis to correctly identify the recombinant variant RF1_2k/1b.
