Using quantitative real-time PCR to determine the hygienic status of cattle manure M. Lebuhn*, M. Effenberger**, A. Gronauer**, P.A. Wilderer* and S. Wuertz* 1 * Lehrstuhl und Versuchsanstalt für Wassergüte- und Abfallwirtschaft, Am Coulombwall, 85748 Garching, Germany (E-mail: m.lebuhn@bv.tum.de) ** Bayerische Landesanstalt für Landtechnik, Am Staudengarten 3, 85354 Freising, Germany 1 Current affiliation: Department of Civil and Environmental Engineering, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA Abstract We developed a suitable system of DNA extraction and real-time quantitative polymerase chain reaction (qPCR) for the specific and sensitive quantification of pathogens and other relevant (indicator) organisms in recalcitrant material such as cattle manure. PCR inhibition by coextraction of humic compounds was minimized in this system, resulting in detection sensitivity of one target DNA copy per reaction well. Data from qPCR analysis for Escherichia coli agreed with cultivation based results, but orders of magnitude more fecal enterococci, Enterobacteriaceae and Campylobacter jejuni, were determined by qPCR than by cultivation. These bacteria may have been in a potentially hazardous active but non-cultivable state. The qPCR system is much less time consuming than conventional cultivation, highly specific, can detect non-cultivable organisms, provides high measurement throughput, and is cost attractive. It should be considered as an alternative in various application areas for (prescribed routine) cultivation based assays, e.g. for biosafety and hygiene monitoring. Keywords Anaerobic digestion; DNA extraction; hygienization; pathogens; PCR inhibitors; quantitative real-time PCR Introduction Anaerobic digestion (AD) of organic wastes is being increasingly used in agricultural and waste(water) treatment practices to produce biogas fuel and organic fertilizer. It is aimed at recycling nutrients and turns out to be a profitable alternative to conventional combustion or aerobic treatment. Moreover, AD can contribute to minimize dissipation of fossil energy resources and greenhouse gas emissions. Various AD techniques have been developed to optimize energy yield, minimize digest volume, and avoid sanitary risks if the digest is to be reused e.g. in agriculture as fertilizer (van Lier et al., 2001). Several studies (e.g. Oropeza et al., 2001) have indicated that effi- cient hygienization during AD may only be achieved if a thermophilic (50–60°C) step with guaranteed retention time at this temperature is included in the process. The aspect of hygienization efficiency of AD has gained major interest in Germany. Water protection areas have been enlarged in the recent past, and in certain areas farmers with animal husbandry are confronted with the problem of increasing animal waste pro- duction but at the same time diminishing land area to apply the manure. This is particularly relevant for former water protection zone III areas that were, or will be, given the status water protection zone II, in which storage and application of manure is generally prohibited (BStMLU, 1994; DVGW, 1995; Anon., 1996). However, exceptions from these restric- tions may be made if the material to be applied has undergone a treatment that guarantees sufficient destruction or inactivation of relevant pathogens. According to German ordinances on drinking water, recreational (bathing) water and biological wastes, quantification of pathogens relies on cultivation techniques. Indicator Water Science and Technology Vol 48 No 4 pp 97–103 © IWA Publishing 2003 97