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
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Czech description
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Classification
Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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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
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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