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©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
New real-time-PCR method to identify single point mutations in hepatitis C virus
Qian Chen, Irene Belmonte, Maria Buti, Leonardo Nieto, Damir Garcia-Cehic, Josep Gregori, Celia Perales, Laura Ordeig, Meritxell Llorens, Maria Eugenia Soria, Rafael Esteban, Juan Ignacio Esteban, Francisco Rodriguez-Frias, Josep Quer
Qian Chen, Maria Buti, Damir Garcia-Cehic, Josep Gregori, Celia Perales, Laura Ordeig, Meritxell Llorens, Maria Eugenia Soria, Rafael Esteban, Juan Ignacio Esteban, Josep Quer, Liver Unit, Internal Medicine, Lab. Malalties Hepàtiques, Vall d’Hebron Institut Recerca-Hospital Universitari Vall d’Hebron, 08035 Barcelona, Spain
Qian Chen, Maria Buti, Damir Garcia-Cehic, Josep Gregori, Celia Perales, Rafael Esteban, Juan Ignacio Esteban, Josep Quer, Centro de Investigación Biomédica en Red, Enfermedades Hepáticas y Digestivas, Instituto de Salud Carlos III, 28029 Madrid, Spain
Irene Belmonte, Leonardo Nieto, Francisco Rodriguez-Frias, Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Autonomous University of Barcelona, 08035 Barcelona, Spain
Irene Belmonte, Leonardo Nieto, Francisco Rodriguez-Frias, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas del Instituto de Salud Carlos III, 28029 Madrid, Spain
Qian Chen, Irene Belmonte, Maria Buti, Leonardo Nieto, Damir Garcia-Cehic, Josep Gregori, Celia Perales, Laura Ordeig, Rafael Esteban, Juan Ignacio Esteban, Francisco Rodriguez-Frias, Josep Quer, Universitat Autònoma de Barcelona, 08193 Barcelona, Spain
Author contributions: Chen Q and Belmonte I have performed the technical research and participated in writing the manuscript; Buti M, Nieto L and Esteban JI participated in the acquisition of serum samples; Garcia-Cehic D and Gregori J contributed to develop analytic tools; Buti M, Perales C, Esteban R, Esteban JI, Rodriguez-Frias F and Quer J revised the article critically and significantly improved the content; Rodriguez-Frias F and Quer J designed the research, developed the invention and have analyzed the data and participated in writing the manuscript; all the authors contributed to this manuscript.
Supported by Instituto de Salud Carlos III, No. PI13/00456, No. PI15/00829, No. PI15/00856, and No. PI12/01893 cofinanced by the European Regional Development Fund (ERDF); the Miguel Servet program of the Instituto de Salud Carlos III, No. CP14/00121 cofinanced by the ERDF; Gilead, No. GLD14/00296; and Instituto de Salud Carlos III, CIBERehd (Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas).
Institutional review board statement: The study was reviewed and approved by the Ethical Committee for Clinical Research (CEIC) from Vall d’Hebron Institut of Research - Hospital Universitari Vall d’Hebron.
Informed consent statement: All study participants, or their legal guardian, provided informed written consent prior to study enrollment.
Conflict-of-interest statement: There are no conflicts of interest to report.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Francisco Rodriguez-Frias, PhD, Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Autonomous University of Barcelona, Pg Vall d’Hebron 119-129, 08035 Barcelona, Spain.
frarodri@vhebron.net
Telephone: +34-93-2746897 Fax: +34-93-4894032
Received: July 11, 2016
Peer-review started: July 13, 2016
First decision: August 19, 2016
Revised: August 25, 2016
Accepted: October 10, 2016
Article in press: October 10, 2016
Published online: November 21, 2016
Processing time: 129 Days and 21.5 Hours
AIM
To develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus (HCV).
METHODS
In patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. LightCycler methods, based on real-time PCR with sequence-specific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest.
RESULTS
Using nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10% (mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80K was detected in 14.6% of G1a patients and 0% of G1b in our setting.
CONCLUSION
A fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This technique can be adapted to detect any single point mutation in highly variable genomes.
Core tip: This report describes a new low-cost, fast and highly sensitive technique based on real-time PCR and specific fluorescence resonance energy transfer probe melting curve analysis to identify single-point mutations in a highly variable background (in this study the Q80K resistance-associated mutation in hepatitis C virus - infected patients) for routine use in clinical laboratories. This technique can be adapted to detect any mutation in a highly variable genome.
