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Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel

[Image: see text] Real-time (quantitative) polymerase chain reaction (qPCR) has been widely applied in molecular diagnostics due to its immense sensitivity and specificity. qPCR multiplexing, based either on fluorescent probes or intercalating dyes, greatly expanded PCR capability due to the concurr...

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Autores principales: Zhang, Haoqing, Yan, Zhiqiang, Wang, Xinlu, Gaňová, Martina, Chang, Honglong, Laššáková, Soňa, Korabecna, Marie, Neuzil, Pavel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2021
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8412922/
https://www.ncbi.nlm.nih.gov/pubmed/34497918
http://dx.doi.org/10.1021/acsomega.1c02971
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author Zhang, Haoqing
Yan, Zhiqiang
Wang, Xinlu
Gaňová, Martina
Chang, Honglong
Laššáková, Soňa
Korabecna, Marie
Neuzil, Pavel
author_facet Zhang, Haoqing
Yan, Zhiqiang
Wang, Xinlu
Gaňová, Martina
Chang, Honglong
Laššáková, Soňa
Korabecna, Marie
Neuzil, Pavel
author_sort Zhang, Haoqing
collection PubMed
description [Image: see text] Real-time (quantitative) polymerase chain reaction (qPCR) has been widely applied in molecular diagnostics due to its immense sensitivity and specificity. qPCR multiplexing, based either on fluorescent probes or intercalating dyes, greatly expanded PCR capability due to the concurrent amplification of several deoxyribonucleic acid sequences. However, probe-based multiplexing requires multiple fluorescent channels, while intercalating dye-based multiplexing needs primers to be designed for amplicons having different melting temperatures. Here, we report a single fluorescent channel-based qPCR duplexing method on a model containing the sequence of chromosomes 21 (Chr21) and 18 (Chr18). We combined nonspecific intercalating dye EvaGreen with a 6-carboxyfluorescein (FAM) probe specific to either Chr21 or Chr18. The copy number (cn) of the target linked to the FAM probe could be determined in the entire tested range from the denaturation curve, while the cn of the other one was determined from the difference between the denaturation and elongation curves. We recorded the amplitude of fluorescence at the end of denaturation and elongation steps, thus getting statistical data set to determine the limit of the proposed method in detail in terms of detectable concentration ratios of both targets. The proposed method eliminated the fluorescence overspilling that happened in probe-based qPCR multiplexing and determined the specificity of the PCR product via melting curve analysis. Additionally, we performed and verified our method using a commercial thermal cycler instead of a self-developed system, making it more generally applicable for researchers. This quantitative single-channel duplexing method is an economical substitute for a conventional rather expensive probe-based qPCR requiring different color probes and hardware capable of processing these fluorescent signals.
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spelling pubmed-84129222021-09-07 Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel Zhang, Haoqing Yan, Zhiqiang Wang, Xinlu Gaňová, Martina Chang, Honglong Laššáková, Soňa Korabecna, Marie Neuzil, Pavel ACS Omega [Image: see text] Real-time (quantitative) polymerase chain reaction (qPCR) has been widely applied in molecular diagnostics due to its immense sensitivity and specificity. qPCR multiplexing, based either on fluorescent probes or intercalating dyes, greatly expanded PCR capability due to the concurrent amplification of several deoxyribonucleic acid sequences. However, probe-based multiplexing requires multiple fluorescent channels, while intercalating dye-based multiplexing needs primers to be designed for amplicons having different melting temperatures. Here, we report a single fluorescent channel-based qPCR duplexing method on a model containing the sequence of chromosomes 21 (Chr21) and 18 (Chr18). We combined nonspecific intercalating dye EvaGreen with a 6-carboxyfluorescein (FAM) probe specific to either Chr21 or Chr18. The copy number (cn) of the target linked to the FAM probe could be determined in the entire tested range from the denaturation curve, while the cn of the other one was determined from the difference between the denaturation and elongation curves. We recorded the amplitude of fluorescence at the end of denaturation and elongation steps, thus getting statistical data set to determine the limit of the proposed method in detail in terms of detectable concentration ratios of both targets. The proposed method eliminated the fluorescence overspilling that happened in probe-based qPCR multiplexing and determined the specificity of the PCR product via melting curve analysis. Additionally, we performed and verified our method using a commercial thermal cycler instead of a self-developed system, making it more generally applicable for researchers. This quantitative single-channel duplexing method is an economical substitute for a conventional rather expensive probe-based qPCR requiring different color probes and hardware capable of processing these fluorescent signals. American Chemical Society 2021-08-19 /pmc/articles/PMC8412922/ /pubmed/34497918 http://dx.doi.org/10.1021/acsomega.1c02971 Text en © 2021 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Zhang, Haoqing
Yan, Zhiqiang
Wang, Xinlu
Gaňová, Martina
Chang, Honglong
Laššáková, Soňa
Korabecna, Marie
Neuzil, Pavel
Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel
title Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel
title_full Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel
title_fullStr Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel
title_full_unstemmed Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel
title_short Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel
title_sort determination of advantages and limitations of qpcr duplexing in a single fluorescent channel
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8412922/
https://www.ncbi.nlm.nih.gov/pubmed/34497918
http://dx.doi.org/10.1021/acsomega.1c02971
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