Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes
Abstract In this report we describe Cy5-dUTP labelling of recombinase-polymerase-amplification (RPA) products directly during the amplification process for the first time. Nucleic acid amplification techniques, especially polymerase-chain-reaction as well as various isothermal amplification methods...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2021-10-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-021-99774-z |
| _version_ | 1819022529944092672 |
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| author | Christian Warmt Carolin Kornelia Fenzel Jörg Henkel Frank Fabian Bier |
| author_facet | Christian Warmt Carolin Kornelia Fenzel Jörg Henkel Frank Fabian Bier |
| author_sort | Christian Warmt |
| collection | DOAJ |
| description | Abstract In this report we describe Cy5-dUTP labelling of recombinase-polymerase-amplification (RPA) products directly during the amplification process for the first time. Nucleic acid amplification techniques, especially polymerase-chain-reaction as well as various isothermal amplification methods such as RPA, becomes a promising tool in the detection of pathogens and target specific genes. Actually, RPA even provides more advantages. This isothermal method got popular in point of care diagnostics because of its speed and sensitivity but requires pre-labelled primer or probes for a following detection of the amplicons. To overcome this disadvantages, we performed an labelling of RPA-amplicons with Cy5-dUTP without the need of pre-labelled primers. The amplification results of various multiple antibiotic resistance genes indicating great potential as a flexible and promising tool with high specific and sensitive detection capabilities of the target genes. After the determination of an appropriate rate of 1% Cy5-dUTP and 99% unlabelled dTTP we were able to detect the bla CTX-M15 gene in less than 1.6E−03 ng genomic DNA corresponding to approximately 200 cfu of Escherichia coli cells in only 40 min amplification time. |
| first_indexed | 2024-12-21T04:24:27Z |
| format | Article |
| id | doaj.art-91e1fe2a91bf4cff8ea48850773e134f |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-21T04:24:27Z |
| publishDate | 2021-10-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-91e1fe2a91bf4cff8ea48850773e134f2022-12-21T19:16:05ZengNature PortfolioScientific Reports2045-23222021-10-011111910.1038/s41598-021-99774-zUsing Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genesChristian Warmt0Carolin Kornelia Fenzel1Jörg Henkel2Frank Fabian Bier3Fraunhofer Institute for Cell Therapy and Immunology - Bioanalytics and Bioprocesses (IZI-BB)Fraunhofer Institute for Cell Therapy and Immunology - Bioanalytics and Bioprocesses (IZI-BB)Fraunhofer Institute for Cell Therapy and Immunology - Bioanalytics and Bioprocesses (IZI-BB)Institute for Biochemistry and Biology, University of PotsdamAbstract In this report we describe Cy5-dUTP labelling of recombinase-polymerase-amplification (RPA) products directly during the amplification process for the first time. Nucleic acid amplification techniques, especially polymerase-chain-reaction as well as various isothermal amplification methods such as RPA, becomes a promising tool in the detection of pathogens and target specific genes. Actually, RPA even provides more advantages. This isothermal method got popular in point of care diagnostics because of its speed and sensitivity but requires pre-labelled primer or probes for a following detection of the amplicons. To overcome this disadvantages, we performed an labelling of RPA-amplicons with Cy5-dUTP without the need of pre-labelled primers. The amplification results of various multiple antibiotic resistance genes indicating great potential as a flexible and promising tool with high specific and sensitive detection capabilities of the target genes. After the determination of an appropriate rate of 1% Cy5-dUTP and 99% unlabelled dTTP we were able to detect the bla CTX-M15 gene in less than 1.6E−03 ng genomic DNA corresponding to approximately 200 cfu of Escherichia coli cells in only 40 min amplification time.https://doi.org/10.1038/s41598-021-99774-z |
| spellingShingle | Christian Warmt Carolin Kornelia Fenzel Jörg Henkel Frank Fabian Bier Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes Scientific Reports |
| title | Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes |
| title_full | Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes |
| title_fullStr | Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes |
| title_full_unstemmed | Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes |
| title_short | Using Cy5-dUTP labelling of RPA-amplicons with downstream microarray analysis for the detection of antibiotic resistance genes |
| title_sort | using cy5 dutp labelling of rpa amplicons with downstream microarray analysis for the detection of antibiotic resistance genes |
| url | https://doi.org/10.1038/s41598-021-99774-z |
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