Factors affecting success of PCR amplification of microsatellite loci from otter faeces

Abstract

We investigated the effect of multiple variables on the amplification success rate of microsatellite DNA extracted from faeces of wild Eurasian otters. The success rate was affected by (i) type of sample, with higher success rates in anal jelly samples than faeces, and (ii) temperature, with a negative effect of increased temperature at time of collection. To increase the effectiveness of microsatellite genotyping of otter faeces, we recommend collecting samples in cold months and early in the morning, preferably in a frozen state, and the collection of anal jelly samples, or the jelly part from faeces, whenever possible.

Effects of time and environmental conditions on the quality of DNA extracted from fecal samples for genotyping of wild deer in a warm temperate broad-leaved forest

“…Furthermore, DNA degradation in feces increases with time after defecation, ambient temperature, humidity, and rainfall (Lucchini et al 2002;Hajkova et al 2006;Brinkman et al 2010;Wedrowicz et al 2013). Consequently, careful selection of fecal samples in the field is important for effective and reliable genetic analysis, and knowledge of the DNA degradation process is useful for selecting feces in the field for genetic research.…”

“…These processes have been examined in a limited range of species and habitats as follows: wolf, Canis lupus (Lucchini et al 2002); brush-tailed rock-wallaby, Petrogale penicillata, and red fox, Vulpes vulpes (Piggott 2004); otter, Lutra lutra (Hajkova et al 2006); brown bear, Ursus arctos (Murphy et al 2007); blacktailed deer, Odocoileus hemionus (Brinkman et al 2010); koala, Phascolarctos cinereus (Wedrowicz et al 2013); pygmy rabbit, Brachylagus idahoensis (DeMay et al 2013); and Sonoran pronghorn, Antilocapra americana (Woodruff et al 2015). With continuing growth in the use of fecal samples in research, there is a need for more information to be gathered on additional species in various types of habitat.…”

Comparison of DNA extraction methods for polymerase chain reaction amplification of guanaco (Lama guanicoe) fecal DNA samples

“…= 1; P = 0.38), suggesting that old feces were as efficient as fresh feces in generating PCR products. While feces freshness is usually considered to be an essential determinant of amplification success (Brinkman et al, 2011), other studies in ungulates and otters have failed to relate the DNA amplification success from fecal material with feces age (Hájková et al, 2006;Lampa et al, 2008;Brinkman et al, 2010). In particular, climatic conditions and air moisture may play an important role in DNA preservation.…”

“…DNA extraction from feces, however, is usually more laborious than from other biological material because of the presence of large amounts of polymerase chain reaction (PCR) inhibitors in fecal material (Waits and Paetkau, 2005). In addition, the quality and quantity of fecal DNA is negatively affected by the exposure to local ambient conditions, the presence of digested food items, and natural degradation processes (Hájková et al, 2006). Herbivore feces, in particular, are known to contain larger quantities of PCR inhibitors than carnivore and omnivore feces (Banks et al, 2002), presumably because of the abundance of secondary metabolites derived from plants in herbivore feces; these metabolites can inhibit the PCR process and impede DNA amplification (Weising et al, 2005).…”

“…Species, geographical localization, diet, season, and PCR primers have all been found to influence the success of DNA amplification from fecal material (Hájková et al, 2006). The optimization of specific extraction protocols is thus critical to increase the number of successful amplifications from fecal material and to reach admissible amplification rates (Valière et al, 2007).…”