Digital polymerase chain reaction duplexing method in a single fluorescence channel

Kód výsledku v 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>

Nalezeny alternativní kódy

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

Výsledek na webu

<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>

Jazyk výsledku

angličtina

Název v původním jazyce

Digital polymerase chain reaction duplexing method in a single fluorescence channel

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

Digital polymerase chain reaction duplexing method in a single fluorescence channel

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10600 - Biological sciences

Projekt

<a href="/cs/project/LTACH19005" target="_blank" >LTACH19005: Vysoce přesný systém digitální polymerázové řetězové reakce pro detekci cfDNA pro neinvazivní prenatální testování (NIPT</a><br>

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2023

Kód důvěrnosti údajů

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

Název periodika

Analytica Chimica Acta

ISSN

0003-2670

e-ISSN

1873-4324

Svazek periodika

1238

Číslo periodika v rámci svazku

January

Stát vydavatele periodika

NL - Nizozemsko

Počet stran výsledku

9

Strana od-do

340243

Kód UT WoS článku

000904882600001

EID výsledku v databázi Scopus

2-s2.0-85137302476