Digital polymerase chain reaction duplexing method in a single fluorescence channel

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00064165%3A_____%2F23%3A10453385" target="_blank" >RIV/00064165:_____/23:10453385 - isvavai.cz</a>

Alternative codes found

RIV/00216208:11110/23:10453385 RIV/00216305:26620/23:PU150312

Result on the web

<a href="https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=ax6e4WQVez" target="_blank" >https://verso.is.cuni.cz/pub/verso.fpl?fname=obd_publikace_handle&handle=ax6e4WQVez</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.aca.2022.340243" target="_blank" >10.1016/j.aca.2022.340243</a>

Result language

angličtina

Original language name

Digital polymerase chain reaction duplexing method in a single fluorescence channel

Original language description

The digital polymerase chain reaction (dPCR) technique can quantify specific sequences of deoxyribonucleic acid using either a droplet-based or chip-based system. dPCR duplexing methods in a single fluorescence channel are typically based on the difference in fluorescence amplitude (F) between two targets. The different targets are distinguished from each other by the F-value variation using non-equal probe concentrations or different target lengths. In the present study, we propose a single fluorescence channel-based dPCR duplexing method that combines a specific probe and intercalating dye to increase the difference in F values between the two targets. We selected two sequences, one from chromosome 18 (Chr18) detected only by the intercalating dye EvaGreen and the other from chromosome 21 (Chr21) detected by a combination of a 6-carboxyfluorescein (FAM) probe and EvaGreen. We performed the dPCR protocol and imaged the dPCR chip at room temperature to verify the proposed duplexing method. The result revealed that the difference in F values between Chr18 and Chr21 increased from approximate to 5% to 20% when using the FAM probe for Chr21 compared with the detection of both amplicons using EvaGreen only. The added FAM probe enabled two-target discrimination using a single-color fluorescent chan-nel. We further determined the difference in F values at different temperatures using artificial dPCR images. This proposed method represents a simple option for single fluorescence channel dPCR duplexing, making it suitable for simplified dPCR systems used for point-of-care applications.

Czech name

—

Czech description

—

Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

—

OECD FORD branch

10600 - Biological sciences

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

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2023

Confidentiality

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

Name of the periodical

Analytica Chimica Acta

ISSN

0003-2670

e-ISSN

1873-4324

Volume of the periodical

1238

Issue of the periodical within the volume

January

Country of publishing house

NL - THE KINGDOM OF THE NETHERLANDS

Number of pages

9

Pages from-to

340243

UT code for WoS article

000904882600001

EID of the result in the Scopus database

2-s2.0-85137302476