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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

Published on: November 5, 2020

Sensitive detection of sample interference in environmental qPCR.

Hyatt C Green¹, Katharine G Field

¹Department of Microbiology, Oregon State University, Corvallis, OR 97331, USA.

|May 8, 2012

Summary

Accurate environmental DNA analysis using quantitative PCR (qPCR) is improved by a new spike-and-recovery method. This approach effectively detects PCR inhibition, ensuring reliable water quality monitoring and environmental research outcomes.

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Area of Science:

Environmental Science
Molecular Biology
Analytical Chemistry

Background:

Quantitative PCR (qPCR) is crucial for environmental monitoring, but sample interference can lead to inaccurate results.
Accurate quantification of molecular markers in environmental samples is often hindered by PCR inhibition.

Purpose of the Study:

To develop and validate a novel spike-and-recovery method for detecting PCR inhibition in environmental samples.
To improve the accuracy of quantitative PCR (qPCR) assays in complex environmental matrices.

Main Methods:

A spike-and-recovery approach using a mutant Escherichia coli strain with a mutant GFP gene was employed.
The method was tested in water samples with reduced extraction efficiency, humic acids, and ethanol.
Kinetic outlier detection (KOD) methods were used to analyze qPCR amplification of control and target markers.

Main Results:

The developed method achieved sensitive detection of PCR inhibition at lower concentrations of inhibitors than traditional methods.
Kinetic outlier detection (KOD) correctly identified inhibition in samples with as little as 0.1 ng humic acids or 5% ethanol.
Sigmoidal modeling distinguished PCR inhibition from poor DNA recovery, enabling simultaneous assessment of inhibition and nucleic acid recovery.

Conclusions:

The spike-and-recovery approach coupled with KOD offers a sensitive and reliable way to identify PCR inhibition in environmental qPCR assays.
This method enhances the accuracy of water quality monitoring and is applicable to various environmental research fields using qPCR.
The ability to distinguish inhibition from poor DNA recovery provides a more comprehensive understanding of sample quality.