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Applied and Environmental Microbiology, November 2009, p. 7253-7260, Vol. 75, No. 22
0099-2240/09/$08.00+0 doi:10.1128/AEM.00796-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Accurate Quantification of Microorganisms in PCR-Inhibiting Environmental DNA Extracts by a Novel Internal Amplification Control Approach Using Biotrove OpenArrays 
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R. van Doorn,1,2
M. M. Klerks,1,3*
M. P. E. van Gent-Pelzer,1
A. G. C. L. Speksnijder,4
G. A. Kowalchuk,2,5 and
C. D. Schoen1
Plant Research International B.V., Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands,1 NIOO-Centre for Terrestrial Ecology, P.O. Box 40, 6666 ZG Heteren, The Netherlands,2 Innosieve Diagnostics B.V., Celsiuslaan 5, 5251 ZB Vlijmen, The Netherlands,3 Public Health Laboratory, Cluster Infectious Diseases, Health Service Amsterdam, Nieuwe Achtergracht 100, 1018 WT Amsterdam, The Netherlands,4 Free University of Amsterdam, Institute of Ecological Science, de Boelelaan 1085, 1081 HV Amsterdam, The Netherlands5
Received 8 April 2009/ Accepted 22 September 2009
PCR-based detection assays are prone to inhibition by substances present in environmental samples, thereby potentially leading to inaccurate target quantification or false-negative results. Internal amplification controls (IACs) have been developed to help alleviate this problem but are generally applied in a single concentration, thereby yielding less-than-optimal results across the wide range of microbial gene target concentrations possible in environmental samples (J. Hoorfar, B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, and P. Fach, J. Clin. Microbiol. 42:1863-1868, 2004). Increasing the number of IACs for each quantitative PCR (qPCR) sample individually, however, typically reduces sensitivity and, more importantly, the reliability of quantification. Fortunately, current advances in high-throughput qPCR platforms offer the possibility of multiple reactions for a single sample simultaneously, thereby allowing the implementation of more than one IAC concentration per sample. Here, we describe the development of a novel IAC approach that is specifically designed for the state-of-the-art Biotrove OpenArray platform. Different IAC targets were applied at a range of concentrations, yielding a calibration IAC curve for each individual DNA sample. The developed IACs were optimized, tested, and validated by using more than 5,000 unique qPCR amplifications, allowing accurate quantification of microorganisms when applied to soil DNA extracts containing various levels of PCR-inhibiting compounds. To our knowledge, this is the first study using a suite of IACs at different target concentrations to monitor PCR inhibition across a wide target range, thereby allowing reliable and accurate quantification of microorganisms in PCR-inhibiting DNA extracts. The developed IAC is ideally suited for high-throughput screenings of, for example, ecological and agricultural samples on next-generation qPCR platforms.
* Corresponding author. Mailing address: Innosieve Diagnostics B.V., Celsiuslaan 5, 5251 ZB Vlijmen, The Netherlands. Phone: 31-646717500. Fax: 31-317418094. E-mail: info{at}innosieve.com
Published ahead of print on 2 October 2009.
Supplemental material for this article may be found at http://aem.asm.org/.
Publication 4574 of The Netherlands Institute of Ecology (NIOO-KNAW).
Applied and Environmental Microbiology, November 2009, p. 7253-7260, Vol. 75, No. 22
0099-2240/09/$08.00+0 doi:10.1128/AEM.00796-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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