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...

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Autores principales:	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
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	mediator probe PCR digital PCR multiplex PCR optimization-free colorectal carcinoma standardized universal reporter Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282
Acceso en línea:	https://doaj.org/article/facbddc1b3114a928e035a7c1228b730
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spelling	oai:doaj.org-article:facbddc1b3114a928e035a7c1228b7302021-11-25T17:03:29ZStringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA10.3390/cancers132257422072-6694https://doaj.org/article/facbddc1b3114a928e035a7c1228b7302021-11-01T00:00:00Zhttps://www.mdpi.com/2072-6694/13/22/5742https://doaj.org/toc/2072-6694There 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.Franziska SchlenkerElena KipfMax DeuterInga HöffkesMichael LehnertRoland ZengerleFelix von StettenFlorian SchererJulius WehrleNikolas von BubnoffPeter JuelgTobias HutzenlaubNadine BorstMDPI AGarticlemediator probe PCRdigital PCRmultiplex PCRoptimization-freecolorectal carcinomastandardized universal reporterNeoplasms. Tumors. Oncology. Including cancer and carcinogensRC254-282ENCancers, Vol 13, Iss 5742, p 5742 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	mediator probe PCR digital PCR multiplex PCR optimization-free colorectal carcinoma standardized universal reporter Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282
spellingShingle	mediator probe PCR digital PCR multiplex PCR optimization-free colorectal carcinoma standardized universal reporter Neoplasms. Tumors. Oncology. Including cancer and carcinogens RC254-282 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
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.
format	article
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
title	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_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_sort	stringent base specific and optimization-free multiplex mediator probe ddpcr for the quantification of point mutations in circulating tumor dna
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/facbddc1b3114a928e035a7c1228b730
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Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA

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