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Related Concept Videos

Real Time RT-PCR02:57

Real Time RT-PCR

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
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Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification
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Digital polymerase chain reaction duplexing method in a single fluorescence channel.

Haoqing Zhang1, Soňa Laššáková2, Zhiqiang Yan3

  • 1Department of Microsystem Engineering, School of Mechanical Engineering, Northwestern Polytechnical University, 127 West Youyi Road, Xi'an, Shaanxi, 710072, PR China; The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an, 710049, PR China.

Analytica Chimica Acta
|December 4, 2022
PubMed
Summary

A new digital polymerase chain reaction (dPCR) method enhances single-channel detection by combining a probe and dye. This improves target discrimination for simplified point-of-care applications.

Keywords:
Absolute quantificationDigital polymerase chain reactionDuplexing methodsSingle fluorescence channel duplexing

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Analytical Chemistry

Background:

  • Digital polymerase chain reaction (dPCR) quantifies DNA sequences using droplet or chip-based systems.
  • Current dPCR duplexing in single channels relies on fluorescence amplitude differences, often using varied probe concentrations or target lengths.
  • Distinguishing multiple targets in a single fluorescence channel presents a technical challenge for simplified dPCR systems.

Purpose of the Study:

  • To develop a novel single fluorescence channel dPCR duplexing method.
  • To enhance the fluorescence amplitude difference between two targets using a combination of a specific probe and an intercalating dye.
  • To validate the proposed method for improved two-target discrimination in dPCR.

Main Methods:

  • A dPCR duplexing strategy was designed using a specific 6-carboxyfluorescein (FAM) probe combined with the intercalating dye EvaGreen.
  • Two targets, chromosome 18 (Chr18) and chromosome 21 (Chr21) sequences, were selected for detection.
  • The method was verified by performing dPCR and imaging at room temperature, with further analysis using artificial dPCR images at varying temperatures.

Main Results:

  • The proposed method significantly increased the fluorescence amplitude difference between Chr18 and Chr21 targets from approximately 5% to 20%.
  • This enhanced difference was achieved by using the FAM probe for Chr21 detection alongside EvaGreen, compared to using EvaGreen alone for both targets.
  • The addition of the FAM probe enabled effective two-target discrimination within a single fluorescence channel.

Conclusions:

  • The developed method offers a straightforward approach for single fluorescence channel dPCR duplexing.
  • Combining a specific probe with an intercalating dye effectively increases fluorescence amplitude differences for improved target discrimination.
  • This technique is well-suited for simplifying dPCR systems, particularly for point-of-care diagnostic applications.