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

The result's identifiers

  • Result code in IS VaVaI

    <a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11110%2F21%3A10432622" target="_blank" >RIV/00216208:11110/21:10432622 - isvavai.cz</a>

  • Alternative codes found

    RIV/00064165:_____/21:10432622 RIV/00216305:26620/21:PU141879

  • Result on the web

    <a href="https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=lKGhyXgzVn" target="_blank" >https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=lKGhyXgzVn</a>

  • DOI - Digital Object Identifier

    <a href="http://dx.doi.org/10.1021/acsomega.1c02971" target="_blank" >10.1021/acsomega.1c02971</a>

Alternative languages

  • Result language

    angličtina

  • Original language name

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

  • Original language description

    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.

  • Czech name

  • Czech description

Classification

  • Type

    J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database

  • CEP classification

  • OECD FORD branch

    30101 - Human genetics

Result continuities

  • Project

    <a href="/en/project/LTACH19005" target="_blank" >LTACH19005: High Precision Digital PCR for cfDNA Detection in Noninvasive Prenatal Testing (NIPT) Applications</a><br>

  • Continuities

    P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Others

  • Publication year

    2021

  • Confidentiality

    S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Data specific for result type

  • Name of the periodical

    ACS Omega [online]

  • ISSN

    2470-1343

  • e-ISSN

  • Volume of the periodical

    6

  • Issue of the periodical within the volume

    34

  • Country of publishing house

    US - UNITED STATES

  • Number of pages

    9

  • Pages from-to

    22292-22300

  • UT code for WoS article

    000692901300041

  • EID of the result in the Scopus database

    2-s2.0-85114470514