Evaluation of QIAampR DNA Stool Mini Kit for ecological studies of gut microbiota Mei Li a,b , Jianhua Gong a, * , Michael Cottrill a , Hai Yu a , Cornelis de Lange b , Jeremy Burton c , Edward Topp d a Food Research Program, Agriculture and Agri-Food Canada, Guelph, Ontario, Canada N1G 5C9 b Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 c Lawson Health Research Institute, University of Western Ontario, London, Ontario, Canada N6A 4V2 d Southern Crop Protection Centre, Agriculture and Agri-Food Canada, London, Ontario, Canada N5V 4T3 Received 5 November 2002; received in revised form 10 December 2002; accepted 10 December 2002 Abstract Cell lysis efficiency and the quality of DNA extracts from complex bacterial ecosystems are two major concerns in molecular ecological studies of gut microbiota. In this study, we use PCR-denaturing gradient gel electrophoresis (DGGE) DNA profiling, random cloning and sequence analysis of 16S rRNA genes to compare the QIAampR DNA Stool Mini Kit with the bead beating technique in the preparation of DNA extracts from gut microbiota of pigs. We also developed a washing procedure that can release more than 93% of bacterial cells attached to the gut mucosa. Both the QIAamp kit and bead beating method lysed approximately 95% of bacterial cells. PCR-DGGE DNA profiles of ileal and cecal microbiota from both digesta and mucosa that were generated from the DNA extracts using the two methods were nearly identical. Random cloning and sequence analysis also demonstrated the high quality of DNA extracts using the two methods. Two random clone sets of 16S rRNA genes generated from the DNA extracts had a similar degree of bacterial diversity. Different preparations of DNA extract from a single sample using the QIAamp kit consistently produced similar PCR-DGGE DNA profiles with similarity indexes higher than 99%. Our data suggest the appropriateness of the QIAampR DNA Stool Mini Kit for the studies of gut microbial ecology and the effectiveness of the QIAamp kit in processing multiple samples for cell lysis and DNA extraction. Crown Copyright D 2003 Published by Elsevier Science B.V. All rights reserved. Keywords: QIAampR; DNA extraction; 16S rRNA; Microbiota 1. Introduction The gut microbiota in farm animals has long been of research interest because of its impact on the health and well-being of the host animals, which is relevant to livestock production efficiency, as well as the safety and quality of livestock products. In the past, the gut microbiota has been mainly studied by culture-based methods. These studies have significantly contributed to our understanding on the gut microbiota. However, because of the limits of culture-based methods, such as medial selectivity for readily culturable bacteria and the presence of non-culturable bacteria (Ricke and Pillai, 1999; Theron and Cloete, 2000), our under- standing of the gut microbiota based on these methods 0167-7012/03/$ - see front matter. Crown Copyright D 2003 Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-7012(02)00260-9 * Corresponding author. Tel.: +1-519-829-2400x3107; fax: +1- 519-829-2600. E-mail address: gongj@agr.gc.ca (J. Gong). www.elsevier.com/locate/jmicmeth Journal of Microbiological Methods 54 (2003) 13 – 20