title: The effect of main urine inhibitors on the activity of different DNA polymerases in loop-mediated isothermal amplification.
abstract: The use of rapid amplification methods to detect pathogens in biological samples is mainly limited by the amount of pathogens present in the sample and the presence of inhibiting substances. Inhibitors can affect the amplification efficiency by either binding to the polymerase, interacting with the DNA, or interacting with the polymerase during primer extension. Amplification is performed using DNA polymerase enzymes and even small changes in their activity can influence the sensitivity and robustness of molecular assays Methods: The main purpose of this research was to examine which compounds present in urine inhibit polymerases with strand displacement activity. To quantify the inhibition, we employed quantitative loop-mediated isothermal amplification Results: The authors found that the presence of BSA, Mg 2+, and urea at physiologically relevant concentrations, as well as acidic or alkaline conditions did not affect the activity of any of the tested polymerases. However, addition of salt significantly affected the activity of the tested polymerases.
  
  
  
  These findings may aid in the development of more sensitive, robust, cost effective isothermal amplification based molecular assays suitable for both point-of-care testing and on-site screening of pathogens directly from unprocessed urine which avoid the need for long and tedious DNA purification steps prior to amplification.
authors.affiliation: a Laboratory of Molecular Biotechnology, Institute of Technology, University of Tartu , Tatru , Estonia.
authors.forename: Jekaterina
authors.initials: J
authors.lastname: Jevtuševskaja
authors.affiliation: a Laboratory of Molecular Biotechnology, Institute of Technology, University of Tartu , Tatru , Estonia.
authors.forename: Katrin
authors.initials: K
authors.lastname: Krõlov
authors.affiliation: b SelfD Technologie GmbH , Leipzig , Germany.|c University of Tartu, Institute of Chemistry , Tartu , Estonia.
authors.forename: Indrek
authors.initials: I
authors.lastname: Tulp
authors.affiliation: a Laboratory of Molecular Biotechnology, Institute of Technology, University of Tartu , Tatru , Estonia.|d Department of Neurochemistry , Stockholm University , Stockholm , Sweden.
authors.forename: Ülo
authors.initials: Ü
authors.lastname: Langel
mesh_terms: D002690:Chlamydia Infections / Q000235:genetics* / Q000652:urine*; D002692:Chlamydia trachomatis / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000652:urine*; D005260:Female; D006801:Humans; D008297:Male; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D014556:Urine / Q000382:microbiology*
chemical_list: D004791:Enzyme Inhibitors
keywords: DNA polymerases; LAMP; Urine inhibitors; loop mediated amplification; point of care; urine