Publication detail

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.

Original Title

Digital polymerase chain reaction duplexing method in a single fluorescence channel

Type

journal article in Web of Science

Language

English

Original Abstract

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.

Keywords

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

Authors

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

Released

15. 1. 2023

Publisher

ELSEVIER

Location

AMSTERDAM

ISBN

1873-4324

Periodical

Analytica Chimica Acta

Year of study

1238

Number

1

State

Kingdom of the Netherlands

Pages count

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"
}