Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA
There is an increasing demand for optimization-free multiplex assays to rapidly establish comprehensive target panels for cancer monitoring by liquid biopsy. We present the mediator probe (MP) PCR for the quantification of the seven most frequent point mutations and corresponding wild types (<i&g...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2021-11-01
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| Series: | Cancers |
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| Online Access: | https://www.mdpi.com/2072-6694/13/22/5742 |
| _version_ | 1797510977086816256 |
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| author | Franziska Schlenker Elena Kipf Max Deuter Inga Höffkes Michael Lehnert Roland Zengerle Felix von Stetten Florian Scherer Julius Wehrle Nikolas von Bubnoff Peter Juelg Tobias Hutzenlaub Nadine Borst |
| author_facet | Franziska Schlenker Elena Kipf Max Deuter Inga Höffkes Michael Lehnert Roland Zengerle Felix von Stetten Florian Scherer Julius Wehrle Nikolas von Bubnoff Peter Juelg Tobias Hutzenlaub Nadine Borst |
| author_sort | Franziska Schlenker |
| collection | DOAJ |
| description | There is an increasing demand for optimization-free multiplex assays to rapidly establish comprehensive target panels for cancer monitoring by liquid biopsy. We present the mediator probe (MP) PCR for the quantification of the seven most frequent point mutations and corresponding wild types (<i>KRAS</i> and <i>BRAF</i>) in colorectal carcinoma. Standardized parameters for the digital assay were derived using design of experiments. Without further optimization, the limit of detection (LoD) was determined through spiking experiments with synthetic mutant DNA in human genomic DNA. The limit of blank (LoB) was measured in cfDNA plasma eluates from healthy volunteers. The 2-plex and 4-plex MP ddPCR assays showed a LoB of 0 copies/mL except for 4-plex <i>KRAS</i> G13D (9.82 copies/mL) and 4-plex <i>BRAF</i> V600E (16.29 copies/mL) and allele frequencies of 0.004% ≤ LoD ≤ 0.38% with R<sup>2</sup> ≥ 0.98. The quantification of point mutations in patient plasma eluates (18 patients) during follow-up using the 4-plex MP ddPCR showed a comparable performance to the reference assays. The presented multiplex assays need no laborious optimization, as they use the same concentrations and cycling conditions for all targets. This facilitates assay certification, allows a fast and flexible design process, and is thus easily adaptable for individual patient monitoring. |
| first_indexed | 2024-03-10T05:38:52Z |
| format | Article |
| id | doaj.art-facbddc1b3114a928e035a7c1228b730 |
| institution | Directory Open Access Journal |
| issn | 2072-6694 |
| language | English |
| last_indexed | 2024-03-10T05:38:52Z |
| publishDate | 2021-11-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cancers |
| spelling | doaj.art-facbddc1b3114a928e035a7c1228b7302023-11-22T22:42:39ZengMDPI AGCancers2072-66942021-11-011322574210.3390/cancers13225742Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNAFranziska Schlenker0Elena Kipf1Max Deuter2Inga Höffkes3Michael Lehnert4Roland Zengerle5Felix von Stetten6Florian Scherer7Julius Wehrle8Nikolas von Bubnoff9Peter Juelg10Tobias Hutzenlaub11Nadine Borst12Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, GermanyHahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, GermanyDepartment of Medicine I, Medical Center—University of Freiburg, Faculty of Medicine, University of Freiburg, 79106 Freiburg, GermanyHahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, GermanyHahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, GermanyHahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, GermanyHahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, GermanyDepartment of Medicine I, Medical Center—University of Freiburg, Faculty of Medicine, University of Freiburg, 79106 Freiburg, GermanyDepartment of Medicine I, Medical Center—University of Freiburg, Faculty of Medicine, University of Freiburg, 79106 Freiburg, GermanyDepartment of Medicine I, Medical Center—University of Freiburg, Faculty of Medicine, University of Freiburg, 79106 Freiburg, GermanyHahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, GermanyHahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, GermanyHahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, GermanyThere is an increasing demand for optimization-free multiplex assays to rapidly establish comprehensive target panels for cancer monitoring by liquid biopsy. We present the mediator probe (MP) PCR for the quantification of the seven most frequent point mutations and corresponding wild types (<i>KRAS</i> and <i>BRAF</i>) in colorectal carcinoma. Standardized parameters for the digital assay were derived using design of experiments. Without further optimization, the limit of detection (LoD) was determined through spiking experiments with synthetic mutant DNA in human genomic DNA. The limit of blank (LoB) was measured in cfDNA plasma eluates from healthy volunteers. The 2-plex and 4-plex MP ddPCR assays showed a LoB of 0 copies/mL except for 4-plex <i>KRAS</i> G13D (9.82 copies/mL) and 4-plex <i>BRAF</i> V600E (16.29 copies/mL) and allele frequencies of 0.004% ≤ LoD ≤ 0.38% with R<sup>2</sup> ≥ 0.98. The quantification of point mutations in patient plasma eluates (18 patients) during follow-up using the 4-plex MP ddPCR showed a comparable performance to the reference assays. The presented multiplex assays need no laborious optimization, as they use the same concentrations and cycling conditions for all targets. This facilitates assay certification, allows a fast and flexible design process, and is thus easily adaptable for individual patient monitoring.https://www.mdpi.com/2072-6694/13/22/5742mediator probe PCRdigital PCRmultiplex PCRoptimization-freecolorectal carcinomastandardized universal reporter |
| spellingShingle | Franziska Schlenker Elena Kipf Max Deuter Inga Höffkes Michael Lehnert Roland Zengerle Felix von Stetten Florian Scherer Julius Wehrle Nikolas von Bubnoff Peter Juelg Tobias Hutzenlaub Nadine Borst Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA Cancers mediator probe PCR digital PCR multiplex PCR optimization-free colorectal carcinoma standardized universal reporter |
| title | Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA |
| title_full | Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA |
| title_fullStr | Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA |
| title_full_unstemmed | Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA |
| title_short | Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA |
| title_sort | stringent base specific and optimization free multiplex mediator probe ddpcr for the quantification of point mutations in circulating tumor dna |
| topic | mediator probe PCR digital PCR multiplex PCR optimization-free colorectal carcinoma standardized universal reporter |
| url | https://www.mdpi.com/2072-6694/13/22/5742 |
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