title: Evaluation of revised polymerase chain reaction primers for more inclusive quantification of ammonia-oxidizing archaea and bacteria.
abstract: Ammonia-oxidizing archaea (AOA) and bacteria (AOB) fill key roles in the nitrogen cycle. Thus, well-vetted methods for characterizing their distribution are essential for framing studies of their significance in natural and managed systems. Quantification of the gene coding for one subunit of the ammonia monooxygenase (amoA) by polymerase chain reaction is frequently employed to enumerate the two groups. However, variable amplification of sequence variants comprising this conserved genetic marker for ammonia oxidizers potentially compromises within- and between-system comparisons. We compared the performance of newly designed non-degenerate quantitative polymerase chain reaction primer sets to existing primer sets commonly used to quantify the amoA of AOA and AOB using a collection of plasmids and soil DNA samples. The new AOA primer set provided improved quantification of model mixtures of different amoA sequence variants and increased detection of amoA in DNA recovered from soils. Although both primer sets for the AOB provided similar results for many comparisons, the new primers demonstrated increased detection in environmental application. Thus, the new primer sets should provide a useful complement to primers now commonly used to characterize the environmental distribution of AOA and AOB.
authors.affiliation: Civil and Environmental Engineering, University of Washington, Seattle, WA, 98105, USA.
authors.forename: Kelley A
authors.initials: KA
authors.lastname: Meinhardt
authors.forename: Anthony
authors.initials: A
authors.lastname: Bertagnolli
authors.forename: Manmeet W
authors.initials: MW
authors.lastname: Pannu
authors.forename: Stuart E
authors.initials: SE
authors.lastname: Strand
authors.forename: Sally L
authors.initials: SL
authors.lastname: Brown
authors.forename: David A
authors.initials: DA
authors.lastname: Stahl
mesh_terms: D000641:Ammonia / Q000378:metabolism*; D001105:Archaea / Q000145:classification* / Q000201:enzymology / Q000302:isolation & purification* / Q000378:metabolism; D001419:Bacteria / Q000145:classification* / Q000201:enzymology / Q000302:isolation & purification* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D008828:Microbiological Techniques / Q000379:methods; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D000641:Ammonia; D010088:Oxidoreductases; C045582:ammonia monooxygenase