Evaluation and optimization of microbial DNA extraction from fecal samples of wild Antarctic bird species

Abstract

Introduction: Advances in the development of nucleic acid-based methods have dramatically facilitated studies of host-microbial interactions. Fecal DNA analysis can provide information about the host's microbiota and gastrointestinal pathogen burden. Numerous studies have been conducted in mammals, yet birds are less well studied. Avian fecal DNA extraction has proved challenging, partly due to the mixture of fecal and urinary excretions and the deficiency of optimized protocols. This study presents an evaluation of the performance in avian fecal DNA extraction of six commercial kits from different bird species, focusing on penguins. Material and methods: Six DNA extraction kits were first tested according to the manufacturers' instructions using mallard feces. The kit giving the highest DNA yield was selected for further optimization and evaluation using Antarctic bird feces. Results: Penguin feces constitute a challenging sample type: most of the DNA extraction kits failed to yield acceptable amounts of DNA. The QIAamp cador Pathogen kit (Qiagen) performed the best in the initial investigation. Further optimization of the protocol resulted in good yields of high-quality DNA from seven bird species of different avian orders. Conclusion: This study presents an optimized approach to DNA extraction from challenging avian fecal samples.

Keywords: Antarctica; Aves; DNA extraction; feces; method evaluation and scatology.

Figure 1.

Agarose gel electrophoresis of kit eluates. (a) DNA yields after extraction with four different kits. From left: QIAamp Fast DNA Stool Mini Kit, QIAamp DNA Stool Mini Kit, DNeasy Blood & Tissue Kit and QIAamp cador Pathogen Kit. Faint smears observed in the DNeasy Blood & Tissue kit and the QIAamp cador Pathogen kit lanes. (b) DNA yields after bead beating pretreatment and extraction with QIAamp cador Pathogen kit. L, DNA ladder. S1, S2 and S3, fecal extracts.

Figure 2.

Agarose gel electrophoresis of C. jejuni-specific PCR with mallard eluates. (a) PCR products from DNA samples extracted with four different kits. Top row from left: QIAamp Fast DNA Stool Mini Kit, QIAamp DNA Stool Mini Kit and DNeasy Blood & Tissue Kit (sample S1). Bottom row from left: DNeasy Blood & Tissue Kit (samples S2 and S3) and QIAamp cador Pathogen Kit. Faint bands were detected in samples extracted with DNeasy Blood & Tissue kit and QIAamp cador Pathogen kit (bottom row). (b) Intense bands visible in samples pretreated with combined heat-shock and bead beating and extracted with the QIAamp cador Pathogen kit. L, DNA ladder. S1, S2 and S3, consecutive samples. NC, negative control. PC, positive control.