High-Purity Microbial DNA Recovery from Chicken Caecal Samples for Metagenomic Analysis

Abstract

Background: The chicken caecum harbours a dense and metabolically active microbial community that shapes host nutrition, immunity, and food-borne pathogen carriage. Reliable metagenomic characterisation of this community depends on recovering microbial DNA that is abundant, pure, and structurally intact, a task complicated by PCR inhibitors and mixed Gram-positive/Gram-negative cell walls in caecal matrices. Objective: This study evaluated the performance and consistency of a silica membrane-based extraction workflow for recovering metagenomic DNA from chicken caecal samples across a 42-day production cycle and two feeding regimens. Methods: Caecal contents were collected from 72 broiler chickens on days 7, 21 and 42 of age, representing synbiotic-supplemented (n = 36) and traditionally fed (n = 36) treatment groups. Metagenomic DNA was extracted using the QIAamp Fast DNA Stool Mini Kit with a thermal lysis modification (90 °C, 5 min). Yield and purity were assessed by NanoDrop spectrophotometry (A260/A280), fluorometric quantification (Qubit dsDNA High-Sensitivity assay), and 0.8% agarose gel electrophoresis. Results: Across all 72 samples, the mean DNA concentration by NanoDrop was 868.9 ± 606.4 ng/µL (range 25.7–2978.8 ng/µL; CV = 69.8%), and the mean A260/A280 ratio was 1.889 ± 0.081 (range 1.56–1.99), with 90.3% of samples (65/72) falling within the target purity window of 1.8–2.0. Qubit fluorometric quantification confirmed high double-stranded DNA content, with 36.1% of samples exceeding the upper limit of the high-sensitivity assay range and requiring dilution. Gel electrophoresis showed intact, high-molecular-weight bands with minimal smearing across sample groups. Yield did not differ consistently between feeding regimens or sampling days, and both concentration and purity ratios were non-normally distributed (Shapiro–Wilk, p < 0.001). Conclusion: The QIAamp Fast DNA Stool Mini Kit, combined with a thermal lysis step, reliably recovered abundant, high-purity, structurally intact metagenomic DNA from chicken caecal samples across ages and feeding regimens, supporting its suitability for large-scale avian gut microbiome and 16S rRNA sequencing studies.