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Improving PCR efficiency for accurate quantification of 16S rRNA genes

Lookup NU author(s): Cameron Callbeck, Dr Angela SherryORCiD, Dr Casey Hubert, Emeritus Professor Neil GrayORCiD, Professor Ian Head


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Quantitative real-time PCR is a valuable tool for microbial ecologists. To obtain accurate absolute quantification it is essential that PCR efficiency for pure standards is close to amplification efficiency for test samples. Counter to normal expectation that PCR efficiency might be lower in environmental DNA, due to the presence of PCR inhibitors, we report the counterintuitive observation that PCR efficiency of pure standards can be lower than for environmental DNA. This can lead to overestimation of gene abundances if not corrected. SYBR green-based qPCR assays of 16S rRNA genes targeting Bacteria, Syntrophus and Smithella spp., Marinobacter spp., Methanomicrobiales, Methanosarcinaceae, and Methanosaetaceae in samples from methanogenic crude oil biodegradation enrichments were tested. In five out of the six assays, PCR efficiency was lower with pure standards than with environmental DNA samples. We developed a solution to this problem based on amending pure clone standards with a background of non-target environmental 16S rRNA genes which significantly improved PCR efficiency of standards in the qPCR assays that exhibited this phenomenon. Overall this method of qPCR standard preparation achieved a more reliable and robust quantification of 16S rRNA genes. We believe this may be a potentially common issue in microbial ecology that often goes unreported, as intuitively one would not expect standards to have poorer PCR efficiency than samples.

Publication metadata

Author(s): Callbeck CM, Sherry A, Hubert CRJ, Gray ND, Voordouw G, Head IM

Publication type: Article

Publication status: Published

Journal: Journal of Microbiological Methods

Year: 2013

Volume: 93

Issue: 2

Pages: 148-152

Print publication date: 01/05/2013

Online publication date: 22/03/2013

Acceptance date: 10/03/2013

ISSN (print): 0167-7012

ISSN (electronic): 1872-8359

Publisher: Elsevier


DOI: 10.1016/j.mimet.2013.03.010


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Funder referenceFunder name
Alberta Innovates-Energy and Environment Solutions
Baker Hughes Inc.
Commercial Microbiology Limited (Intertek)
Computer Modelling Group Limited
Industrial Research Chair Award
Shell Canada Limited
Suncor Energy Developments Inc.
Yara International ASA
Aramco Services
2007-1Federation of European Microbiological Societies (FEMS Research Fellowship)
NE/E01657X/1Natural Environment Research Council