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New real-time-PCR method to identify single point mutations in hepatitis C virus

World Journal of Gastroenterology 2016年第43期22卷 9604-9612页

作者： 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 Liver Unit Internal Medicine Lab. Malalties Hepàtiques Vall d’Hebron Institut Recerca-Hospital Universitari Vall d’ Hebron08035 Barcelona Spain Centro de Investigación Biomédica en Red Enfermedades Hepáticas y Digestivas Instituto de Salud Carlos Ⅲ28029 madrid Spain Liver Pathology Unit Departments of Biochemistry and Microbiology Hospital Universitari Vall d’Hebron Autonomous University of Barcelona 08035 Bareelona Spain Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas del Instituto de Salud Carlos Ⅲ 28029 Madrid Spain Universitat Autònoma de Barcelona 08193 Bareelona Spain

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 Q80 K 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. Light Cycler methods, based on real-time PCR with sequencespecific 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 Q80 K 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. Q80 K was detected in 14.6% of G1 a patients and 0% of G1 b 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

关键词： Hepatitis C virus Resistance-associated amino acid substitutions Low-cost test Single-point mutations Q80K

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Effect of the manipulation of the duodenal papilla during double balloon enteroscopy

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World Journal of Gastroenterology 2016年第17期22卷 4330-4337页

作者： Rafael Latorre Octavio López-Albors Federico soria eugenia Candanosa Enrique Pérez-Cuadrado Department of Anatomy and Comparative Pathology Veterinary Faculty University of Murcia Campus Universitario de Espinardo 30100 Murcia Spain Minimally Invasive Surgery Center Jesus Uson 10071 Caceres Spain Unit of Digestive Diseases Morales Meseguer University Hospital 30008 Murcia Spain

AIM: To determine the hypothesis that inflating the balloons in the duodenal papilla determines changes in the biochemical markers of ***: Four groups of pigs were used: Group papilla(GP), the overtube's balloon was inflated in the area of the papilla; GP + double balloon enteroscopy(GP + DBE), the overtube's balloon was kept inflated in the area of the papilla for 20 min before a DBE; Group DBE(GDBE), DBE was carried out after insuring the balloon's inflation far from the pancreatic papilla; and Group control(GC). Serum concentrations of amylase, lipase and C-reactive protein(CRP) were evaluated. Pancreases were processed for histopathology ***: Main changes occurred 24 h after the procedure compared with baseline levels. Amylase levels increased significantly in GP(59.2% higher) and were moderately higher in groups GP + DBE and GDBE(22.7% and 20%, respectively). Lipase increased in GP and GP + DBE, whereas it hardly changed in GDBE and in GC. CRP increased significantly in GP, GP + DBE and GDBE, while no changes were reported for GC. No statistically significant difference between groups GP and GP + DBE was found for the histopathological findings, except for vacuolization and necrosis of the pancreatic parenchyma that was higher in GP than in GP + ***: The manipulation of the duodenal papilla by the inflated overtube's balloon during DBE causes pancreatic structural damage and increased biochemical markers associated with pancreatitis.

关键词： Duodenal papilla Double balloon enteroscopy Pancreas Animal model Pancreatitis

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