Comparison of DNA Extraction Methods for Molecular Detection of Probiotic Lactobacilli, Lysis-resistant Bacteria

Abstract

DNA extraction is a crucial step in all nucleic acid-based protocols to identify microorganisms. Lactic acid bacteria are a significant part of healthy microbiota in the human gastrointestinal tract. These gram-positive bacteria have several layers of peptidoglycan in the cell walls. These structures cause difficulties in the cell lysis and obtaining reliable protocols for DNA isolations. The purpose of this study was to assess the autoclave and lysozyme-based DNA purification approaches for achieving the high-quality genomic DNAs of Lactobacillus acidophilus bacteria. DNA concentrations and qualities were also compared with the commercial kit. The results showed that the proper DNA isolation methods were various, according to the downstream applications. Protocols that included lysozyme produced a higher amount of DNA than the autoclave method. Lysozyme treatment combined with silica-guanidinethiocyanate procedure was the efficient protocol with affordable cost for routine lysis of L. acidophilus bacteria. Appropriate DNA concentration and quality were obtained through this method comparable to those of the commercial kit. Inversely, autoclave treatment had little effect on the breakage of the cell walls indicating low concentrations of extracted DNAs. This method could not completely break down all the bacterial cell walls. However, the breakage of low numbers of cell walls was microscopically observed in the supernatant of the autoclaved cell suspension. The quality of this protocol was found to be adequate for performing direct polymerase chain reaction (PCR) assay on samples with large amounts of lactobacilli. These conclusions suggest attentively selecting the DNA extraction method based on the planned downstream analysis of PCR products.

Keywords

• autoclave
• direct pcr
• dna purification
• lactobacillus acidophilus
• lysozyme