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1 July 2015 A simple and effective method for obtaining mammal DNA from faeces
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Abstract

The use of faecal DNA, although a promising tool for the population monitoring of mammals, has not yet become a fully exploited and standard practice, mainly because low target DNA concentration, DNA degradation, and co-purification of inhibitors demand extra laboratory procedures to improve success and reliability. Here we evaluate a simple method that enables sampling of DNA in the field through the collection of the intestinal cells present on the surface of a scat using a swab. The swab is immediately placed in a vial containing a lysis buffer that preserves the DNA for its later extraction. DNA extracts of three species of herbivores (goat, fallow deer and white-tailed deer), two carnivores (Iberian lynx and domestic dog) and one omnivore species (brushtail possum) were characterised in terms of target and total DNA quantity, PCR inhibition and genotyping success. Direct comparison was carried out with duplicate samples preserved in 96% ethanol and extracted via a commonly used commercial DNA extraction kit for faecal material. Results from these comparisons show that swabbing the samples in situ not only simplifies field collection and sample handling in the laboratory, but generally optimises target DNA recovery, minimises co-purification of PCR inhibitors and provides good quality DNA for the species tested, especially for herbivores. This method is also less time-consuming and more cost-effective, thus providing a more convenient and efficient alternative for non-invasive genetic studies.

Non-invasive samples, and particularly faeces, provide potential sources of genetic information for ecological and demographic surveys. Faecal material can yield information of presence, abundance, genetic diversity, relatedness, phylogeography, sex and dispersal (Schwartz and Monfort 2008, see Beja-Pereira et al. 2009 for a comprehensive review of non-invasive genetic sampling). Faecal material as a DNA source has many advantages, such as 1) non-invasiveness; meaning there is no need for handling or even locating the animals; 2) availability; faeces are constantly generated by all individuals; and 3) relatively easy detection; faeces are normally the most obvious remnants from scarce or elusive animals and can be detected by trained dogs (McKay et al. 2008).

However, there are significant limitations to the routine use of faecal DNA. These include scarcity and degradation of the DNA and the co-purification of PCR inhibitors. These factors dramatically reduce the genotyping success of faecal samples and impose the need for pilot optimisation analyses and multiple genotyping replicates, which overall increase the cost of such studies (Fernando et al. 2003). These limitations have historically made faecal DNA either impracticable or unaffordable. Despite an increase in non-invasive DNA usage in general (Taberlet et al. 1999), and faecal DNA in particular since the 1990s (Frantz et al. 2003, Rutledge et al. 2009, Caryl et al. 2012, Ebert et al. 2012), prospects for its extensive application to population monitoring have remained unfulfilled. Since all the above limitations are ultimately intrinsic to the sample, finding more efficient methods of collection, preservation and DNA extraction that are viable in the field and laboratory should widen the use of faecal DNA for population monitoring and genetic studies, and eventually facilitate its use in the expanding field of genomics (Perry et al. 2010).

Faeces usually carry PCR inhibitors from the soil, diet contents or the digestive system. Problems caused by coextracted inhibitors, together with DNA degradation and low template DNA concentrations, range from amplification failure to allelic dropout and peak imbalance (Opel et al. 2010). Removal of inhibitors normally involves an extra step of incubation with materials such as InhibitEX tablets (Qiagen, Hilden, Germany) or starch (Zhang et al. 2006, Kawamoto et al. 2013). Alternatively, partial inhibition can be circumvented by diluting the extract and increasing the number of cycles in the PCR, although this can result in higher rates of amplification failure and genotyping errors (Fernando et al. 2003). Inhibition is thus a major source of genotyping failure and can substantially increase genotyping costs. Consequently, methods that minimize inhibitor carryover should be favoured in surveys based on faecal DNA.

Most of the stool DNA is from an exogenous origin, i.e. non-target DNA from microbes, diet or coprophagous animals (Bradley and Vigilant 2002); whereas the endogenous DNA (target DNA in this study), originated from sloughed epithelial cells of the intestine wall (Fernando et al. 2003), constitutes a minor proportion of the total faecal DNA (Perry et al. 2010). Since epithelial cells are supposed to accumulate in the outer layer of the scat, several sampling strategies have targeted the enrichment of these cells by peeling off, scraping or washing the outer layer of the scat before DNA extraction. However, whole or large portions of scats are still commonly sampled in the field (Ball et al. 2007, Cullingham et al. 2010, Reddy et al. 2012). (See Eggert et al. 2005 and Tende et al. 2014 for reviews on faecal preservation methods). Unfortunately, molecular ecology sampling campaigns often take place in remote areas under severe conditions that do complicate these collection procedures, and impose transport and storage limitations (Camacho-Sanchez et al. 2013). Alternatively, a swab can be used directly in the field to recover the outer layer of faeces enriched for sloughed intestinal cells, thereby avoiding the need for storing and transporting bulky pieces of faeces in ethanol, silica beads or as frozen samples (Rutledge et al. 2009). Although the swabbing sampling protocol, ex situ or in situ, has proven effective for a few species (Ball et al. 2007, Rutledge et al. 2009, Cullingham et al. 2010, Renan et al. 2012), no cross-species studies have formally validated whether the added simplicity impacts on DNA recovery, inhibition rates and subsequent PCR amplification efficiency.

The aim of this study is to validate a standardised simple field collection and automated high-throughput extraction protocol for faecal DNA that has potential for widespread application. The simple method of swabbing the faecal samples directly in the field, i.e. swabbing in situ, immediate pr