Observational Study
Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Nov 21, 2016; 22(43): 9604-9612
Published online Nov 21, 2016. doi: 10.3748/wjg.v22.i43.9604
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
Core Tip

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.