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.

Diagnostic reliability of nested PCR depends on the primer design and threshold abundance ofHelicobacter pyloriin biopsy, stool, and saliva samples

“…Consequently, the most plausible explanation is that the nested PCR assay has similar sensitivity to 13 Mini Kit, which is widely used for this purpose and is specifically designed to remove these inhibitors. 51 We often observed amplification inhibition in the first PCR reaction, especially when the DNA volume exceeded the standard 0.5 µL. Nevertheless, the inhibition was not profound in the spiking experiments.…”

How should we store avian faecal samples for microbiota analyses? Comparing efficacy and cost-effectiveness

“…Faecal sampling is commonly used for representing intestinal microbiota because it is non-invasive. Yet obtaining reliable molecular data from avian faeces is complicated by its chemical composition, as digestive excreta is mixed with urinary products such as uric acid that can degrade DNA or interfere with DNA extraction (Eriksson et al, 2017;Regnaut et al, 2006). The result is that DNA yields from avian faeces are typically low, making amplification difficult and pipelines more sensitive to contamination.…”

Cloacal swabs and alcohol bird specimens are good proxies for compositional analyses of gut microbial communities of Great tits (Parus major)

“…Although we did not compare fecal samples with cloacal swabs, the bacterial taxa we acquired from cloacal swabs were similar to taxa characterized in fecal microbiomes of wild P. major in a previous study [16], suggesting that microbiome discrepancies between these sampling methods are minor. Furthermore, the cloacal swab approach also reduces the risk of contamination and removes the problems associated with the typically lower DNA yield from extractions of bird feces [42,43].…”