Conventional versus real-time quantitative PCR for rare species detection

Area of Science:

• Environmental DNA (eDNA) analysis
• Molecular ecology
• Invasive species detection

Background:

• Detecting rare species in natural environments is challenging.
• Conventional PCR (cPCR) and real-time quantitative PCR (qPCR) are common for eDNA detection.
• A direct comparison of cPCR and qPCR for eDNA detection is lacking, hindering optimal method selection.

Purpose of the Study:

• To comprehensively compare the detection capacity and false negative rates of cPCR and qPCR.
• To evaluate these methods for detecting the invasive golden mussel (Limnoperna fortunei) in various sample types.
• To provide guidance on selecting the most effective PCR method for rare species detection using eDNA.

Main Methods:

• Comparison of cPCR and qPCR using DNA samples from laboratory aquaria and field irrigation channels.
• Testing of limit of detection (LoD), limit of quantification (LoQ), detection rate, and false negative rate.
• Analysis of eDNA concentration relative to distance from the source and potential PCR inhibition in environmental samples.

Main Results:

• qPCR exhibited a lower LoD (1 × 10⁻⁷ ng/μl) than cPCR (10⁻⁶ ng/μl).
• qPCR achieved higher detection rates: 100% for lab samples and 68.6% for field samples, versus 87.9% and 47.1% for cPCR, respectively.
• Field samples showed evidence of inhibition, requiring higher target DNA concentrations for quantification. False negative rates decreased with increased sample replication.

Conclusions:

• qPCR is more effective than cPCR for detecting rare species using eDNA, especially in complex environmental samples.
• The higher sensitivity and detection rates of qPCR make it the preferred method for rare species monitoring.
• Minimizing false negatives requires extensive sampling and consideration of potential inhibitors in field eDNA studies.