Quantitative analysis of ammonia oxidising bacteria using competitive PCR

Area of Science:

• Environmental microbiology
• Molecular biology

Background:

• Culture-based methods like most probable number (MPN) are inefficient for enumerating ammonia-oxidizing bacteria due to isolation and cultivation challenges.
• Laboratory-cultivated bacteria may not accurately represent environmental populations, introducing bias in traditional enumeration.

Purpose of the Study:

• To develop and validate a competitive PCR (cPCR)-based method for enumerating beta-subgroup proteobacterial ammonia-oxidizing bacteria.
• To compare the accuracy of cPCR with traditional MPN methods for quantifying these bacteria in agricultural soils and estuarine sediments.

Main Methods:

• Development of a cPCR assay targeting 16S rRNA genes specific to beta-subgroup proteobacterial ammonia oxidizers.
• Quantification of ammonia oxidizer populations in agricultural soils and estuarine sediments using both MPN and cPCR techniques.
• Construction of separate standard curves for each sample type due to variations in DNA extraction, quantity, and purity impacting PCR efficiency.

Main Results:

• MPN methodologies significantly underestimated ammonia oxidizer populations, yielding counts 1-3 orders of magnitude lower than cPCR.
• cPCR detected higher ammonia oxidizer numbers in fertilized agricultural soils, a difference not observed with MPN.
• Sample-specific standard curves were crucial for accurate cPCR quantification due to variations in environmental DNA.

Conclusions:

• Competitive PCR offers a more sensitive and accurate method for enumerating ammonia-oxidizing bacteria compared to traditional culture-based approaches.
• This improved quantification reveals higher bacterial abundances in environmental samples, particularly in agricultural soils.
• The cPCR method provides a valuable tool for understanding microbial ecology and the impact of environmental factors on bacterial populations.