Abstract
The use of non-invasive genetic sampling (NGS) is becoming increasingly important in the study of wild animal populations. Obtaining DNA from faecal samples is of particular interest because faeces can be collected without deploying sample capture devices. However, PCR amplification of DNA extracted from faeces is problematic because of high concentrations of inhibitors. Here we present a method for increasing the successful application of donor DNA extracted from faecal samples through inhibitor reduction. After standard extraction with a DNA stool kit we used a ‘Concentrated Chelex Treatment’ (CCT) that increased the amplification success from 31.7 to 61.4% of loci. Our results suggest that darker supernatant and samples with more precipitate contain more inhibitors than lighter samples and samples with little or no precipitate. We expect the use of this technique to have wide applicability within conservation biology for research and management that relies on NGS of wild animal populations.
References
Creel S, Spong G, Sands JL, Rotella J, Zeigle J, Joe L, Murphy KM, Smith D (2003) Population size estimation in Yellowstone wolves with error-prone noninvasive microsatellite genotypes. Mol Ecol 12:2003–2009
Frantz AC, Pope LC, Carpenter PJ, Roper TJ, Wilson GJ, Delahay RJ, Burke T (2003) Reliable microsatellite genotyping of the eurasian badger (Meles meles) using faecal DNA. Mol Ecol 12:1649–1661
Fredholm M, Winterø AK (1995) Variation of short tandem repeats within and between species belonging to the canidae family. Mamm Genome 6:11–18
Gonzalez S, Maldonado JE, Ortega J, Talarico AC, Bidegaray-Batista L, Garcia JE, Duarte JMB (2009) Identification of the endangered small red brocket deer (Mazama bororo) using noninvasive genetic techniques (Mammalia; Cervidae). Mol Ecol 9:754–758
Hedmark E, Flagstad Ø, Segerström P, Persson J, Landa A, Ellegren H (2004) DNA-based individual and sex identification from wolverine (Gulo gulo) faeces and urine. Conserv Genet 5:405–410
Idaghdour Y, Broderick D, Korrida A (2003) Faeces as a source of DNA for molecular studies in a threatened population of great bustards. Conserv Genet 4:789–792
Kitchen AM, Gese EM, Waits LP, Karki SM, Schauster ER (2005) Genetic and spatial structure within a swift fox population. J Anim Ecol 74:1173–1181
Lantz PG, Matsson M, Wadstrom T, Radstrom P (1997) Removal of PCR inhibitors from human faecal samples through the use of an aqueous two-phase system for sample preparation prior to PCR. J Microbiol Methods 28:159–167
McKelvey KS, Schwartz MK (2004) Genetic errors associated with population estimation using non-invasive molecular tagging: problems and new solutions. J Wildl Manag 68:439–448
Monteiro L, Bonnemaison D, Vekris A, Petry KG, Bonnet J, Vidal R, Cabrita J, Megraud F (1997) Complex polysaccharides as PCR inhibitors in feces: helicobacter pylori model. J Clin Microbiol 35:995–998
Pérez T, Vázquez F, Naves J, Fernández A, Corao A, Albornoz J, Domínguez A (2008) Non-invasive genetic study of the endangered cantabrian brown bear (Ursus arctos). Conserv Genet 10:291–301
Quéméré E, Louis Jr EE, Ribéron A, Chikhi L, Cronuau-Roy B (2009) Non-invasive conservation genetics of the critically endangered golden-crowned sifaka (Propithecus tattersalli): high diversity and significant genetic differentiation over a small range. Conserv Gene. doi:10.1007/s10592-009-9837-9
Smith DA, Ralls K, Hurt A, Adams B, Parker M, Maldonado JE (2006) Assessing reliability of microsatellite genotypes from kit fox faecal samples using genetic and GIS analyses. Mol Ecol 15:387–406
Waits LP, Paetkau D (2005) Noninvasive genetic sampling tools for wildlife biologists: a review of applications and recommendations for accurate data collection. J Wildl Manag 69:1419–1433
Acknowledgments
We would like to acknowledge the assistance of M. Leibst-Olsen, P.C. Lieck, S. Mathiesen and M. Strunge in carrying out this work. Funding was provided by Framework Grants (No. 21-04-0403 and 272-07-0477) from the Danish Council for Independent Research/Natural Sciences to T.D. and an Oticon Foundation grant to L.H.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hebert, L., Darden, S.K., Pedersen, B.V. et al. Increased DNA amplification success of non-invasive genetic samples by successful removal of inhibitors from faecal samples collected in the field. Conservation Genet Resour 3, 41–43 (2011). https://doi.org/10.1007/s12686-010-9280-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12686-010-9280-8