Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients

Abstract Circulating cell-free DNA (cfDNA) has been investigated as a screening tool for many diseases. To avoid expensive and time-consuming DNA isolation, direct quantification PCR assays can be established. However, rigorous validation is required to provide reliable data in the clinical and non-...

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Autores principales:	Elmo W. I. Neuberger, Alexandra Brahmer, Tobias Ehlert, Katrin Kluge, Keito F. A. Philippi, Simone C. Boedecker, Julia Weinmann-Menke, Perikles Simon
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/2c8625c02e284cb487322ebbc35471f4
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spelling	oai:doaj.org-article:2c8625c02e284cb487322ebbc35471f42021-12-02T16:31:57ZValidating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients10.1038/s41598-021-92826-42045-2322https://doaj.org/article/2c8625c02e284cb487322ebbc35471f42021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-92826-4https://doaj.org/toc/2045-2322Abstract Circulating cell-free DNA (cfDNA) has been investigated as a screening tool for many diseases. To avoid expensive and time-consuming DNA isolation, direct quantification PCR assays can be established. However, rigorous validation is required to provide reliable data in the clinical and non-clinical context. Considering the International Organization for Standardization, as well as bioanalytical method validation guidelines, we provide a comprehensive procedure to validate assays for cfDNA quantification from blood plasma without DNA isolation. A 90 and 222 bp assay was validated to study the kinetics of cfDNA after exercise in patients with systemic lupus erythematosus (SLE). The assays showed ultra-low limit of quantification (LOQ) with 0.47 and 0.69 ng/ml, repeatability ≤ 11.6% (95% CI 8.1–20.3), and intermediate precision ≤ 12.1% (95% CI 9.2–17.7). Incurred sample reanalysis confirmed the precision of the procedure. The additional consideration of pre-analytical factors shows that centrifugation speed and temperature do not change cfDNA concentrations. In SLE patients cfDNA increases ~ twofold after a walking exercise, normalizing after 60 min of rest. The established assays allow reliable and cost-efficient quantification of cfDNA in minute amounts of plasma in the clinical setting. Additionally, the assay can be used as a tool to determine the impact of pre-analytical factors and validate cfDNA quantity and quality of isolated samples.Elmo W. I. NeubergerAlexandra BrahmerTobias EhlertKatrin KlugeKeito F. A. PhilippiSimone C. BoedeckerJulia Weinmann-MenkePerikles SimonNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Elmo W. I. Neuberger Alexandra Brahmer Tobias Ehlert Katrin Kluge Keito F. A. Philippi Simone C. Boedecker Julia Weinmann-Menke Perikles Simon Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients
description	Abstract Circulating cell-free DNA (cfDNA) has been investigated as a screening tool for many diseases. To avoid expensive and time-consuming DNA isolation, direct quantification PCR assays can be established. However, rigorous validation is required to provide reliable data in the clinical and non-clinical context. Considering the International Organization for Standardization, as well as bioanalytical method validation guidelines, we provide a comprehensive procedure to validate assays for cfDNA quantification from blood plasma without DNA isolation. A 90 and 222 bp assay was validated to study the kinetics of cfDNA after exercise in patients with systemic lupus erythematosus (SLE). The assays showed ultra-low limit of quantification (LOQ) with 0.47 and 0.69 ng/ml, repeatability ≤ 11.6% (95% CI 8.1–20.3), and intermediate precision ≤ 12.1% (95% CI 9.2–17.7). Incurred sample reanalysis confirmed the precision of the procedure. The additional consideration of pre-analytical factors shows that centrifugation speed and temperature do not change cfDNA concentrations. In SLE patients cfDNA increases ~ twofold after a walking exercise, normalizing after 60 min of rest. The established assays allow reliable and cost-efficient quantification of cfDNA in minute amounts of plasma in the clinical setting. Additionally, the assay can be used as a tool to determine the impact of pre-analytical factors and validate cfDNA quantity and quality of isolated samples.
format	article
author	Elmo W. I. Neuberger Alexandra Brahmer Tobias Ehlert Katrin Kluge Keito F. A. Philippi Simone C. Boedecker Julia Weinmann-Menke Perikles Simon
author_facet	Elmo W. I. Neuberger Alexandra Brahmer Tobias Ehlert Katrin Kluge Keito F. A. Philippi Simone C. Boedecker Julia Weinmann-Menke Perikles Simon
author_sort	Elmo W. I. Neuberger
title	Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients
title_short	Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients
title_full	Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients
title_fullStr	Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients
title_full_unstemmed	Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients
title_sort	validating quantitative pcr assays for cfdna detection without dna extraction in exercising sle patients
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/2c8625c02e284cb487322ebbc35471f4
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Validating quantitative PCR assays for cfDNA detection without DNA extraction in exercising SLE patients

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