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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.
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
URL: http://dx.doi.org/10.1016/j.mimet.2013.03.010
DOI: 10.1016/j.mimet.2013.03.010
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