Open AccessQuantitative analysis of ammonia oxidising bacteria using competitive PCR
Author(s) -
Phillips Carol J.,
Paul Eldor A.,
Prosser James I.
Publication year - 2000
Publication title -
fems microbiology ecology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.377
H-Index - 155
eISSN - 1574-6941
pISSN - 0168-6496
DOI - 10.1111/j.1574-6941.2000.tb00710.x
Subject(s) - biology , bacteria , enumeration , most probable number , 16s ribosomal rna , isolation (microbiology) , ammonia , polymerase chain reaction , microbiology and biotechnology , gene , genetics , biochemistry , mathematics , combinatorics
Culture‐based methods for enumeration, such as most probable number (MPN) methodologies, have proved inefficient due to difficulties in the isolation and cultivation of ammonia oxidising bacteria in the laboratory. Biases are associated with the isolation of bacteria in selective media and organisms cultivated in the laboratory may not be truly representative of those in the environment. In this study, we developed a competitive PCR (cPCR)‐based method based on the amplification of 16S rRNA genes specific for the β‐subgroup proteobacterial ammonia oxidising bacteria for enumeration of these organisms. Populations in both agricultural soils and estuarine sediments were quantified by traditional MPN and by cPCR. The numbers of ammonia oxidisers for both sample types were significantly underestimated by conventional MPN and were 1–3 orders of magnitude lower than those obtained by cPCR. Higher numbers of ammonia oxidisers found in fertilised plots in agricultural soils by the cPCR technique were not observed in MPN estimates. It was necessary to construct a separate standard curve for each sample type as differences in DNA extraction, quantity and purity had a significant bearing on the ease of PCR of both competitor and target DNA.