Choice of DNA extraction method affects detection of bacterial taxa from retail chicken breast

• Published in: BMC microbiology 2022-09, Vol.22 (1), p.1-230, Article 230
• Main Authors: Flint, Annika, Laidlaw, Anna, Li, Leo, Raitt, Courtney, Rao, Mary, Cooper, Ashley, Weedmark, Kelly, Carrillo, Catherine, Tamber, Sandeep
• Format: Article
• Language: English
• Subjects: 16S rRNA amplicon sequencing; Bacteria; Bacterial culture; Biotyper; Chickens; Culture independent detection; Deoxyribonucleic acid; DNA; DNA extraction; DNA sequencing; Food; Food quality; Food safety; Gene amplification; Gene sequencing; Genetic testing; Identification; Laboratories; Lysis; Metagenomics; Methods; Microbiomes; Microbiota (Symbiotic organisms); Pathogens; Polymerase chain reaction; Poultry; Reagents; Relative abundance; RNA sequencing; rRNA 16S; Taxa; Testing
• ISSN: 1471-2180; 1471-2180
• DOI: 10.1186/s12866-022-02650-7

Sequence-based methods for the detection of bacteria such as 16S rRNA amplicon sequencing and metagenomics can provide a comprehensive view of the bacterial microbiome of food. These methods rely on the detection of gene sequences to indicate the presence of viable bacteria. This indirect form of detection can be prone to experimental artefacts. Sample handling and processing are key sources of variation that require standard approaches. Extracting sufficient quantities of high quality DNA from food matrices is challenging because target bacterial species are usually minor components of the microbiota and foods contain an array of compounds that are inhibitory to downstream DNA applications. Here, three DNA extraction methods are compared for their ability to extract high quality bacterial DNA from retail chicken breast rinses, with or without enrichment. Method performance was assessed by comparing ease of use, DNA yield, DNA quality, PCR amplicon yield, and the detection of bacterial taxa by 16S rRNA amplicon sequencing. All three DNA extraction methods yielded DNA of sufficient quantity and quality to perform quantitative PCR and 16S rRNA amplicon sequencing. The extraction methods differed in ease of use, with the two commercial kits (PowerFood, PowerSoil) offering considerable time and cost savings over a hybrid method that used laboratory reagents for lysis and commercial column based kits for further purification. Bacterial richness as determined by 16S rRNA amplicon sequencing was similar across the three DNA extraction methods. However, differences were noted in the relative abundance of bacterial taxa, with significantly higher abundance of Gram-positive genera detected in the DNA samples prepared using the PowerFood DNA extraction kit. The choice of DNA extraction method can affect the detection of bacterial taxa by 16S rRNA amplicon sequencing in chicken meat rinses. Investigators should be aware of this procedural bias and select methods that are fit for the purposes of their investigation.