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Real Time RT-PCR

Real Time RT-PCR

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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Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

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Quantifying the Detection Sensitivity and Precision of qPCR and ddPCR Mechanisms for eDNA Samples.

Gledis Guri^1,2, Jessica Louise Ray³, Andrew Olaf Shelton⁴

¹Norwegian Institute of Marine Research Framsenteret Tromsø Norway.

Ecology and Evolution

|December 13, 2024

Summary

Droplet digital PCR (ddPCR) shows higher sensitivity and precision than quantitative PCR (qPCR) for environmental DNA (eDNA) detection, especially at low concentrations. This research guides improved aquatic species monitoring methods using ddPCR.

Keywords:

ddPCR eDNA fish monitoring qPCR quantification precision sensitivity

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Area of Science:

Environmental DNA (eDNA) analysis
Aquatic biodiversity monitoring
Molecular ecology

Background:

Environmental DNA (eDNA) offers a non-invasive method for aquatic organism monitoring.
Accurate eDNA quantification is vital for tracking rare/invasive species and biodiversity.
Quantitative PCR (qPCR) and droplet digital PCR (ddPCR) are key eDNA detection techniques.

Purpose of the Study:

To compare the sensitivity and quantification precision of qPCR and ddPCR for eDNA surveys.
To evaluate these methods across three teleost fish species assays.
To guide researchers toward more reliable eDNA detection methods.

Main Methods:

Bayesian inference was used to analyze latent parameters from known concentrations (standards) and environmental samples.
Comparison of qPCR and ddPCR performance in detecting and quantifying eDNA.
Investigation of the lower limit of quantification for ddPCR and its applicability to qPCR.

Main Results:

ddPCR demonstrated superior sensitivity and quantification precision compared to qPCR, particularly at low DNA concentrations (< 1 copy/μL reaction).
A two-step model (detection and concentration) improved qPCR precision for eDNA quantification.
Insights into extending the lower limit of quantification for ddPCR were provided.

Conclusions:

ddPCR is a more sensitive and precise method for eDNA detection at low concentrations.
Findings support the use of ddPCR for more reliable aquatic species monitoring.
Methodological improvements, like the two-step model for qPCR, can enhance eDNA quantification robustness.