PCR enrichment techniques to identify the diet of predators

Abstract

The increasing sensitivity of PCR has meant that in the last two decades PCR has emerged as a major tool in diet studies, enabling us to refine our understanding of trophic links and to elucidate the diets of predators whose prey is as yet uncharacterized. The achievements and methods of PCR-based diet studies have been reviewed several times, but here we review an important development in the field: the use of PCR enrichment techniques to promote the amplification of prey DNA over that of the predator. We first discuss the success of using group-specific primers either in parallel single reactions or in multiplex reactions. We then concentrate on the more recent use of PCR enrichment techniques such as restriction enzyme digests, peptide nucleic acid clamping, DNA blocking and laser capture microdissection. We also survey the vast literature on enrichment techniques in clinical biology, to ascertain the pitfalls of enrichment techniques and what refinements have yielded some highly sensitive methods. We find that while there are several new approaches to enrichment, peptide nucleic acid clamping and DNA blocking are generally sufficient techniques for the characterization of diets of predators and highlight the most important considerations of the approach.

The use of DNA barcodes in food web construction—terrestrial and aquatic ecologists unite!

“…What is evident is that the number of studies using DNA barcodes in constructing nodes and links in aquatic food webs is currently increasing, and that the development of methods is picking up speed (e.g., for recent papers of methods involving blocking primers, see O'Rorke et al 2012a;Leray et al 2013; for improved quantifications, see Thomas et al 2014). Thus, we expect the next few years to bring studies where DNA barcodes have been applied simultaneously to resolve the nodes and the links of "full" aquatic food webs (or substantial parts of them).…”

Specific and sensitive primers for the detection of predated olive fruit flies, Bactrocera oleae (Diptera: Tephritidae)

“…The specificity and sensitivity tests revealed that all primer combinations were appropriate for molecular detection of predation under laboratory conditions (O'Rorke et al, 2012). However, it was also necessary to test their reliability under real conditions of digestion.…”

“…The reliable identification of a prey species requires that only prey DNA sequences are detected. The design of species-specific primers is therefore essential (O'Rorke et al, 2012). Since digestion of the prey results in degradation of its DNA, the mitochondrial cytochrome oxidase I (COI) gene appears as a suitable target for amplification because there are several copies of the gene per organelle and also several organelles per cell.…”

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