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Quantitative Estimation of Low-Abundance Targets in Engineered Systems and Environmental Samples: Comparative Study
Alessia Ayala Alban1, Barbara Tonanzi1,2, Simona Crognale1,2
1Water Research Institute, National Research Council of Italy, CNR-IRSA, Via Salaria Km 29.300, Monterotondo, 00015 Rome, Italy.
Droplet digital PCR (ddPCR) offers superior sensitivity and precision for detecting low-abundance ammonia-oxidizing bacteria (AOB) in complex environmental samples compared to quantitative PCR (qPCR). This advancement is crucial for environmental monitoring and wastewater treatment processes.
Area of Science:
- Environmental Microbiology
- Molecular Biology
- Biotechnology
Background:
- Quantitative PCR (qPCR) is widely used for nucleic acid detection but is limited by external calibration and inhibition in environmental samples.
- Droplet digital PCR (ddPCR) excels in low-abundance target quantification, yet its application in complex environmental matrices is less explored.
- Ammonia-oxidizing bacteria (AOB) are key players in nitrogen cycling and wastewater treatment, necessitating accurate quantification methods.
Purpose of the Study:
- To compare the performance of ddPCR and qPCR for quantifying ammonia-oxidizing bacteria (AOB) in diverse environmental and engineered samples.
- To evaluate the sensitivity and reproducibility of both methods in the presence of PCR inhibitors and low target concentrations.
- To assess the suitability of ddPCR for complex samples with low target-to-non-target ratios.
Main Methods:
- Comparative analysis of ddPCR and qPCR assays using Eva/SYBR Green and TaqMan chemistries.
- Serial dilutions of DNA were tested across seven different environmental and engineered sample types.
- Focus on quantifying ammonia-oxidizing bacteria (AOB) as the target analyte.
Main Results:
- ddPCR consistently produced precise, reproducible, and statistically significant results across all tested samples.
- ddPCR demonstrated enhanced sensitivity in detecting AOB in complex samples with low target abundance and low target/non-target ratios.
- qPCR performance was potentially limited by sample complexity and the presence of inhibitors, unlike ddPCR.
Conclusions:
- ddPCR is a highly sensitive and reliable method for quantifying AOB in challenging environmental and engineered samples.
- ddPCR overcomes limitations of qPCR, particularly in low-abundance scenarios and complex matrices.
- The findings support the adoption of ddPCR for accurate microbial monitoring in environmental and wastewater treatment applications.
