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Direct DNA and RNA detection from large volumes of whole human blood

PCR inhibitors in clinical specimens negatively affect the sensitivity of diagnostic PCR and RT-PCR or may even cause false-negative results. To overcome PCR inhibition, increase the sensitivity of the assays and simplify the detection protocols, simple methods based on quantitative nested real-time...

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Autores principales: Cai, Dongyang, Behrmann, Ole, Hufert, Frank, Dame, Gregory, Urban, Gerald
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821888/
https://www.ncbi.nlm.nih.gov/pubmed/29467420
http://dx.doi.org/10.1038/s41598-018-21224-0
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author Cai, Dongyang
Behrmann, Ole
Hufert, Frank
Dame, Gregory
Urban, Gerald
author_facet Cai, Dongyang
Behrmann, Ole
Hufert, Frank
Dame, Gregory
Urban, Gerald
author_sort Cai, Dongyang
collection PubMed
description PCR inhibitors in clinical specimens negatively affect the sensitivity of diagnostic PCR and RT-PCR or may even cause false-negative results. To overcome PCR inhibition, increase the sensitivity of the assays and simplify the detection protocols, simple methods based on quantitative nested real-time PCR and RT-PCR were developed to detect exogenous DNA and RNA directly from large volumes of whole human blood (WHB). Thermus thermophilus (Tth) polymerase is resistant to several common PCR inhibitors and exhibits reverse transcriptase activity in the presence of manganese ions. In combination with optimized concentrations of magnesium ions and manganese ions, Tth polymerase enabled efficient detection of DNA and RNA from large volumes of WHB treated with various anticoagulants. The applicability of these methods was further demonstrated by examining WHB specimens collected from different healthy individuals and those stored under a variety of conditions. The detection limit of these methods was determined by detecting exogenous DNA, RNA, and bacteria spiked in WHB. To the best of our knowledge, direct RNA detection from large volumes of WHB has not been reported. The results of the developed methods can be obtained within 4 hours, making them possible for rapid and accurate detection of disease-causing agents from WHB.
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spelling pubmed-58218882018-02-26 Direct DNA and RNA detection from large volumes of whole human blood Cai, Dongyang Behrmann, Ole Hufert, Frank Dame, Gregory Urban, Gerald Sci Rep Article PCR inhibitors in clinical specimens negatively affect the sensitivity of diagnostic PCR and RT-PCR or may even cause false-negative results. To overcome PCR inhibition, increase the sensitivity of the assays and simplify the detection protocols, simple methods based on quantitative nested real-time PCR and RT-PCR were developed to detect exogenous DNA and RNA directly from large volumes of whole human blood (WHB). Thermus thermophilus (Tth) polymerase is resistant to several common PCR inhibitors and exhibits reverse transcriptase activity in the presence of manganese ions. In combination with optimized concentrations of magnesium ions and manganese ions, Tth polymerase enabled efficient detection of DNA and RNA from large volumes of WHB treated with various anticoagulants. The applicability of these methods was further demonstrated by examining WHB specimens collected from different healthy individuals and those stored under a variety of conditions. The detection limit of these methods was determined by detecting exogenous DNA, RNA, and bacteria spiked in WHB. To the best of our knowledge, direct RNA detection from large volumes of WHB has not been reported. The results of the developed methods can be obtained within 4 hours, making them possible for rapid and accurate detection of disease-causing agents from WHB. Nature Publishing Group UK 2018-02-21 /pmc/articles/PMC5821888/ /pubmed/29467420 http://dx.doi.org/10.1038/s41598-018-21224-0 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Cai, Dongyang
Behrmann, Ole
Hufert, Frank
Dame, Gregory
Urban, Gerald
Direct DNA and RNA detection from large volumes of whole human blood
title Direct DNA and RNA detection from large volumes of whole human blood
title_full Direct DNA and RNA detection from large volumes of whole human blood
title_fullStr Direct DNA and RNA detection from large volumes of whole human blood
title_full_unstemmed Direct DNA and RNA detection from large volumes of whole human blood
title_short Direct DNA and RNA detection from large volumes of whole human blood
title_sort direct dna and rna detection from large volumes of whole human blood
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821888/
https://www.ncbi.nlm.nih.gov/pubmed/29467420
http://dx.doi.org/10.1038/s41598-018-21224-0
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