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The effect of main urine inhibitors on the activity of different DNA polymerases in loop-mediated isothermal amplification
Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
Number of Authors: 4
2017 (English)In: Expert Review of Molecular Diagnostics, ISSN 1473-7159, E-ISSN 1744-8352, Vol. 17, no 4, 403-410 p.Article, review/survey (Refereed) Published
Abstract [en]

Background: 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 assaysMethods: 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 amplificationResults: 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.Conclusion: 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.

Place, publisher, year, edition, pages
2017. Vol. 17, no 4, 403-410 p.
Keyword [en]
Urine inhibitors, loop mediated amplification, LAMP, DNA polymerases, urine, point of care
National Category
Biological Sciences Chemical Sciences Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-143617DOI: 10.1080/14737159.2017.1283218ISI: 000399478700009OAI: oai:DiVA.org:su-143617DiVA: diva2:1103338
Available from: 2017-05-30 Created: 2017-05-30 Last updated: 2017-05-30Bibliographically approved

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