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Agreement between amoA Gene-Specific Quantitative PCR and Fluorescence In Situ Hybridization in the Measurement of Ammonia-Oxidizing Bacteria in Activated Sludge

Lookup NU author(s): Dr Joana Baptista, Professor Russell DavenportORCiD, Donna Swan, Fiona Read, Dr Mathew Brown, Cristiana Morais, Professor Thomas CurtisORCiD


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Microbial abundance is central to most investigations in microbial ecology, and its accurate measurement is a challenging task that has been significantly facilitated by the advent of molecular techniques over the last 20 years. Fluorescence in situ hybridization (FISH) is considered the gold standard of quantification techniques; however, it is expensive and offers low sample throughput, both of which limit its wider application. Quantitative PCR (qPCR) is an alternative that offers significantly higher throughput, and it is used extensively in molecular biology. The accuracy of qPCR can be compromised by biases in the DNA extraction and amplification steps. In this study, we compared the accuracy of these two established quantification techniques to measure the abundance of a key functional group in biological wastewater treatment systems, the ammonia-oxidizing bacteria (AOB), in samples from a time-series experiment monitoring a set of laboratory-scale reactors and a full-scale plant. For the qPCR analysis, we tested two different sets of AOB-specific primers, one targeting the 16SrRNA gene and one targeting the ammonia monooxygenase (amoA) gene. We found that there was a positive linear logarithmic relationship between FISH and the amoA genespecific qPCR, where the data obtained from both techniques was equivalent at the order of magnitude level. The 16S rRNA gene-specific qPCR assay consistently underestimated AOB numbers.

Publication metadata

Author(s): Baptista JDC, Lunn M, Davenport RJ, Swan DL, Read LF, Brown MR, Morais C, Curtis TP

Publication type: Article

Publication status: Published

Journal: Applied and Environmental Microbiology

Year: 2014

Volume: 80

Issue: 19

Pages: 5901-5910

Print publication date: 01/10/2014

Online publication date: 07/07/2014

Acceptance date: 30/06/2014

ISSN (print): 0099-2240

ISSN (electronic): 1098-5336

Publisher: American Society for Microbiology


DOI: 10.1128/AEM.01383-14


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Funder referenceFunder name
EP/H012133/1Engineering and Physical Sciences Research Council