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

Simple item page
dc.contributor.authorZhang, Haoqingcs
dc.contributor.authorYan, Zhiqiangcs
dc.contributor.authorWang, Xinlucs
dc.contributor.authorGaňová, Martinacs
dc.contributor.authorChang, Honglongcs
dc.contributor.authorLaššáková, Soňacs
dc.contributor.authorNeužil, Pavelcs
dc.contributor.authorKorabečná, Mariecs
dc.coverage.issue34cs
dc.coverage.volume6cs
dc.date.accessioned2021-12-06T06:55:39Z
dc.date.available2021-12-06T06:55:39Z
dc.date.issued2021-08-31cs
dc.description.abstractReal-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.en
dc.formattextcs
dc.format.extent22292-22300cs
dc.format.mimetypeapplication/pdfcs
dc.identifier.citationACS OMEGA. 2021, vol. 6, issue 34, p. 22292-22300.en
dc.identifier.doi10.1021/acsomega.1c02971cs
dc.identifier.issn2470-1343cs
dc.identifier.other172879cs
dc.identifier.urihttp://hdl.handle.net/11012/203053
dc.language.isoencs
dc.publisherAmerican Chemical Societycs
dc.relation.ispartofACS OMEGAcs
dc.relation.urihttps://pubs.acs.org/doi/10.1021/acsomega.1c02971cs
dc.rightsCreative Commons Attribution-NonCommercial-NoDerivatives 4.0 Internationalcs
dc.rights.accessopenAccesscs
dc.rights.sherpahttp://www.sherpa.ac.uk/romeo/issn/2470-1343/cs
dc.rights.urihttp://creativecommons.org/licenses/by-nc-nd/4.0/cs
dc.subjectreal-time PCRen
dc.subjectquantitative PCRen
dc.subjecthigh-throughputen
dc.subjectamplificationen
dc.titleDetermination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channelen
dc.type.driverarticleen
dc.type.statusPeer-revieweden
dc.type.versionpublishedVersionen
sync.item.dbidVAV-172879en
sync.item.dbtypeVAVen
sync.item.insts2021.12.06 07:55:39en
sync.item.modts2021.12.03 12:12:40en
thesis.grantorVysoké učení technické v Brně. Středoevropský technologický institut VUT. Chytré nanonástrojecs
thesis.grantorVysoké učení technické v Brně. Fakulta elektrotechniky a komunikačních technologií. Ústav mikroelektronikycs
Files
Original bundle
Now showing 1 - 1 of 1
Thumbnail Image
Name:
acsomega.1c02971.pdf
Size:
3.1 MB
Format:
Adobe Portable Document Format
Description:
acsomega.1c02971.pdf
Download
Collections
Ústav mikroelektroniky
Chytré nanonástroje