Development of a real-time PCR method for quantification of the three genera Dehalobacter , Dehalococcoides, and Desulfitobacterium in microbial communities Theo H.M. Smits, Christiane Devenoges, Katia Szynalski, Julien Maillard, Christof Holliger * Swiss Federal Institute of Technology (EPFL), ENAC-Laboratory for Environmental Biotechnology, Ba ˆtiment Chimie CH-B Ecublens, CH-1015 Lausanne, Switzerland Received 17 December 2003; received in revised form 6 February 2004; accepted 10 February 2004 Available online 14 March 2004 Abstract We developed standard curves based on plasmids containing a 16S rRNA gene of a member of one of the three genera Dehalobacter, Desulfitobacterium, and Dehalococcoides. A large difference in amplification efficiency between the standard curves was observed ranging from 1.5 to 2.0. The total eubacterial 16S rRNA gene copy number determined in a sample DNA by using eubacterial primers and the three standard curves led to differences in the estimated copy numbers of a factor up to 73. However, the amplification efficiencies for one specific standard curve were the same independent of the PCR primer pair used. This allowed the determination of the abundance of a population expressed as fractional number, hence, the percentage of genus-specific copy numbers within the total eubacterial 16S rRNA gene copy numbers. Determination of the fractional numbers in DNA mixtures of known composition showed the accuracy of this approach. The average difference in threshold value between two 10-fold dilutions of DNA of pure cultures, mixtures thereof and of environmental samples was  3.45 F 0.34, corresponding to an average almost optimal amplification efficiency of 1.95. This indicated that the low amplification efficiency of certain standard curves seemed to be mainly a problem of the plasmid DNA used and not of the 16S rRNA gene of the target genera. D 2004 Elsevier B.V. All rights reserved. Keywords: Real-time PCR; Population abudance; Microbial communities 1. Introduction Chloroethenes have been entering the environment as a result of improper handling, storage or spillage. Due to their low degradation potential under aerobic conditions, they can seep into groundwater. There, degradation under anaerobic conditions, either by oxidation under iron- or manganese-reducing condi- tions (Bradley and Chapelle, 1998) or by complete reductive dechlorination to ethene (Holliger et al., 1999), can take place. Reductive dechlorination is considered as the major process for the biodegradation 0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2004.02.003 * Corresponding author. Tel.: +41-21-693-47-24; fax: +41-21- 693-47-22. E-mail address: christof.holliger@epfl.ch (C. Holliger). www.elsevier.com/locate/jmicmeth Journal of Microbiological Methods 57 (2004) 369 – 378