Shwani, A.; Zuo, B.; Alrubaye, A.; Zhao, J.; Rhoads, D.D. A Simple, Inexpensive Alkaline Method for Bacterial DNA Extraction from Environmental Samples for PCR Surveillance and Microbiome Analyses. Appl. Sci.2024, 14, 141.
Shwani, A.; Zuo, B.; Alrubaye, A.; Zhao, J.; Rhoads, D.D. A Simple, Inexpensive Alkaline Method for Bacterial DNA Extraction from Environmental Samples for PCR Surveillance and Microbiome Analyses. Appl. Sci. 2024, 14, 141.
Shwani, A.; Zuo, B.; Alrubaye, A.; Zhao, J.; Rhoads, D.D. A Simple, Inexpensive Alkaline Method for Bacterial DNA Extraction from Environmental Samples for PCR Surveillance and Microbiome Analyses. Appl. Sci.2024, 14, 141.
Shwani, A.; Zuo, B.; Alrubaye, A.; Zhao, J.; Rhoads, D.D. A Simple, Inexpensive Alkaline Method for Bacterial DNA Extraction from Environmental Samples for PCR Surveillance and Microbiome Analyses. Appl. Sci. 2024, 14, 141.
Abstract
DNA extraction for downstream molecular diagnostic applications can be an expensive, time-consuming process. We devised a method to quickly extract genomic DNA from environmental samples based on sodium hydroxide lysis of cells with or without capture by magnetic beads, for subsequent PCR or quantitative PCR. The final DNA extraction method using NaOH is extremely low-cost and can be completed in 10 minutes at room temperature. NaOH extraction was effective for Gram-negative and Gram-positive bacteria in samples from air, soil, sewage, food, laboratory surfaces, and chicken cloacal swabs. The NaOH extraction method was comparable to commercial kits for extraction of DNA from pig fecal samples for 16S amplicon sequencing analyses. We demonstrate that an impinger and portable pump can efficiently capture bacteria in poultry facilities for rapid DNA extraction for quantification of total bacteria and for detection of specific species using qPCR. The air sampling and NaOH extraction procedures are well-suited for routine, high throughput screening, and for metagenomic analyses for specific pathogens, even in resource-limited situations.
Keywords
microbiome; bacteria; DNA extraction; poultry; swine; air sampling
Subject
Biology and Life Sciences, Biology and Biotechnology
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
