Here, we report the electrochemical detection of single-point mutations using solid-phase isothermal primer elongation with redox-labeled oligonucleotides. A single-base mutation associated with resistance to rifampicin, an antibiotic commonly used for the treatment of Mycobacterium tuberculosis, was used as a model system to demonstrate a proof-of-concept of the approach. Four 5'-thiolated primers, designed to be complementary with the same fragment of the target sequence and differing only in the last base, addressing the polymorphic site, were self-assembled via chemisorption on individual gold electrodes of an array. Following hybridization with single-stranded DNA, Klenow (exo-) DNA polymerase-mediated primer extension with ferrocene-labeled 2'-deoxyribonucleoside triphosphates (dNFcTPs) was only observed to proceed at the electrode where there was full complementarity between the surface-tethered probe and the target DNA being interrogated. We tested all four ferrocenylethynyl-linked dNTPs and optimized the ratio of labeled/natural nucleotides to achieve maximum sensitivity. Following a 20 min hybridization step, Klenow (exo-) DNA polymerase-mediated primer elongation at 37 °C for 5 min was optimal for the enzymatic incorporation of a ferrocene-labeled nucleotide, achieving unequivocal electrochemical detection of a single-point mutation in 14 samples of genomic DNA extracted from Mycobacterium tuberculosis strains. The approach is rapid, cost-effective, facile, and can be extended to multiplexed electrochemical single-point mutation genotyping.

• Klíčová slova
• Klenow (exo-) DNA polymerase, ferrocene-labeled nucleotides, nucleoside triphosphates, single-nucleotide polymorphism (SNP), single-point mutation, solid-phase primer elongation,
• MeSH
• jednonukleotidový polymorfismus MeSH
• metaloceny MeSH
• Mycobacterium tuberculosis * genetika   MeSH
• oxidace-redukce MeSH
• rifampin farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• metaloceny MeSH
• rifampin MeSH

Phage therapy is increasingly put forward as a "new" potential tool in the fight against antibiotic resistant infections. During the "Centennial Celebration of Bacteriophage Research" conference in Tbilisi, Georgia on 26-29 June 2017, an international group of phage researchers committed to elaborate an expert opinion on three contentious phage therapy related issues that are hampering clinical progress in the field of phage therapy. This paper explores and discusses bacterial phage resistance, phage training and the presence of prophages in bacterial production strains while reviewing relevant research findings and experiences. Our purpose is to inform phage therapy stakeholders such as policy makers, officials of the competent authorities for medicines, phage researchers and phage producers, and members of the pharmaceutical industry. This brief also points out potential avenues for future phage therapy research and development as it specifically addresses those overarching questions that currently call for attention whenever phages go into purification processes for application.

• Klíčová slova
• Bacteriophage, adaptation, phage therapy, production, prophage, regulation, resistance,
• MeSH
• Bacteria genetika   virologie   MeSH
• bakteriální infekce mikrobiologie   terapie   MeSH
• bakteriofágy fyziologie   MeSH
• fágová terapie * metody   MeSH
• lidé MeSH
• mikrobiologie životního prostředí MeSH
• potravinářská mikrobiologie MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• kongresy MeSH
• práce podpořená grantem MeSH