Improving PCR detection of prey in molecular diet studies: importance of group‐specific primer set selection and extraction protocol performances

Abstract

While the morphological identification of prey remains in predators' faeces is the most commonly used method to study trophic interactions, many studies indicate that this method does not detect all consumed prey. Polymerase chain reaction-based methods are increasingly used to detect prey DNA in the predator food bolus and have proven efficient, delivering highly accurate results. When studying complex diet samples, the extraction of total DNA is a critical step, as polymerase chain reaction (PCR) inhibitors may be co-extracted. Another critical step involves a careful selection of suitable group-specific primer sets that should only amplify DNA from the targeted prey taxon. In this study, the food boluses of five Rattus rattus and seven Rattus exulans were analysed using both morphological and molecular methods. We tested a panel of 31 PCR primer pairs targeting bird, invertebrate and plant sequences; four of them were selected to be used as group-specific primer pairs in PCR protocols. The performances of four DNA extraction protocols (QIAamp(®) DNA stool mini kit, DNeasy(®) mericon food kit and two of cetyltrimethylammonium bromide-based methods) were compared using four variables: DNA concentration, A(260) /A(280) absorbance ratio, food compartment analysed (stomach or faecal contents) and total number of prey-specific PCR amplification per sample. Our results clearly indicate that the A(260) /A(280) absorbance ratio, which varies between extraction protocols, is positively correlated to the number of PCR amplifications of each prey taxon. We recommend using the DNeasy(®) mericon food kit (QIAGEN), which yielded results very similar to those achieved with the morphological approach.

Diet analysis of small mammal pests: A comparison of molecular and microhistological methods

“…Furthermore, prey type and its amount in the sample can also affect DNA-based detection. For example, prey present in trace amounts or composed of keratinous and chitinous cells could be recognizable in microhistological analysis, but DNA extraction from such material would be difficult (Zarzoso-Lacoste et al 2013). Our results agree with this statement, as plant species that remain undetected in DNA analysis were occurring in a relatively small amount in the contents (Table 3).…”

“…This was not an issue with R. rattus as the group-specific primers used in the present study had been previously tested for specificity (Zarzoso-Lacoste et al 2013). However, in some instances with M. m. castaneus and S. murinus host DNA was detected with the invertebrate primer set (C1J1718/C1N2191).…”

Clearing muddied waters: Capture of environmental DNA from turbid waters

“…Though the DNeasy mericon Food kit has not yet been tested on eDNA to our knowledge, this kit performed well with fecal samples that likely also contained inhibitors prior to extraction [61]. Both protocols use CTAB which can either form complexes with nucleic acids in low salt conditions or with cellular inhibitors in high salt conditions.…”

History, applications, methodological issues and perspectives for the use environmental DNA (eDNA) in marine and freshwater environments

“…The direct observation or identification of the prey in the stomach or in the fecal material often is difficult and can reduce the taxonomic resolution or introduce bias (Braley, Goldsworthy, Page, Steer, & Austin, 2010). Thus, the use of eDNA fragments or metabarcoding approach using stomach contents as target DNA can be used for dietary and trophic studies without the observation or identification of the prey in the stomach or feces (Zarzoso-Lacoste, Corse, & vidal, 2013). 8.…”