Detail publikace

Digital polymerase chain reaction duplexing method in a single fluorescence channel

ZHANG, H. LAŠŠÁKOVÁ, S. YAN, Z. WANG, X. ŠENKYŘÍK, P. GAŇOVÁ, M. CHANG, H. KORABEČNÁ, M. NEUŽIL, P.

Originální název

Digital polymerase chain reaction duplexing method in a single fluorescence channel

Typ

článek v časopise ve Web of Science, Jimp

Jazyk

angličtina

Originální abstrakt

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.

Klíčová slova

Digital polymerase chain reaction; Absolute quantification; Duplexing methods; Single fluorescence channel duplexing

Autoři

ZHANG, H.; LAŠŠÁKOVÁ, S.; YAN, Z.; WANG, X.; ŠENKYŘÍK, P.; GAŇOVÁ, M.; CHANG, H.; KORABEČNÁ, M.; NEUŽIL, P.

Vydáno

15. 1. 2023

Nakladatel

ELSEVIER

Místo

AMSTERDAM

ISSN

1873-4324

Periodikum

Analytica Chimica Acta

Ročník

1238

Číslo

1

Stát

Nizozemsko

Strany počet

9

URL

https://www.sciencedirect.com/science/article/pii/S0003267022008145?via%3Dihub

BibTex

@article{BUT187277,
  author="Haoqing {Zhang} and Soňa {Laššáková} and Zhiqiang {Yan} and Xinlu {Wang} and Pavel {Šenkyřík} and Martina {Gaňová} and Honglong {Chang} and Marie {Korabečná} and Pavel {Neužil}",
  title="Digital polymerase chain reaction duplexing method in a single fluorescence channel",
  journal="Analytica Chimica Acta",
  year="2023",
  volume="1238",
  number="1",
  pages="9",
  doi="10.1016/j.aca.2022.340243",
  issn="1873-4324",
  url="https://www.sciencedirect.com/science/article/pii/S0003267022008145?via%3Dihub"
}