title: The yield and quality of cellular and bacterial DNA extracts from human oral rinse samples are variably affected by the cell lysis methodology.
abstract: Recent culture-independent studies have enabled detailed mapping of human microbiome that has not been hitherto achievable by culture-based methods. DNA extraction is a key element of bacterial culture-independent studies that critically impacts on the outcome of the detected microbial profile. Despite the variations in DNA extraction methods described in the literature, no standardized technique is available for the purpose of microbiome profiling. Hence, standardization of DNA extraction methods is urgently needed to yield comparable data from different studies. We examined the effect of eight different cell lysis protocols on the yield and quality of the extracted DNA from oral rinse samples. These samples were exposed to cell lysis techniques based on enzymatic, mechanical, and a combination of enzymatic-mechanical methods. The outcome measures evaluated were total bacterial population, Firmicutes levels and human DNA contamination (in terms of surrogate GAPDH levels). We noted that all three parameters were significantly affected by the method of cell lysis employed. Although the highest yield of gDNA was obtained using lysozyme-achromopeptidase method, the lysozyme-zirconium beads method yielded the peak quantity of total bacterial DNA and Firmicutes with a lower degree of GAPDH contamination compared with the other methods. Taken together our data clearly points to an urgent need for a consensus, standardized DNA extraction technique to evaluate the oral microbiome using oral rinse samples. Further, if Firmicutes levels are the focus of investigation in oral rinse microbiome analyses then the lysozyme-zirconium bead method would be the method of choice in preference to others.
authors.affiliation: School of Medical Science, Menzies Health Institute Queensland, Griffith University, Gold Coast 4215, Australia.
authors.forename: Mohsen
authors.initials: M
authors.lastname: Sohrabi
authors.affiliation: School of Dentistry and Oral Health, Menzies Health Institute Queensland, Griffith University, Gold Coast 4215, Australia. Electronic address: r.nair@griffith.edu.au.
authors.forename: Raj G
authors.initials: RG
authors.lastname: Nair
authors.affiliation: School of Dentistry, Oral Health Centre, University of Queensland, Brisbane 4072, Australia.
authors.forename: Lakshman P
authors.initials: LP
authors.lastname: Samaranayake
authors.affiliation: Centre for Health Practice Innovation, Menzies Health Institute Queensland, Griffith University, Nathan 4111, Australia.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.affiliation: School of Medical Science, Menzies Health Institute Queensland, Griffith University, Gold Coast 4215, Australia.
authors.forename: Abu Hasanat Md
authors.initials: AH
authors.lastname: Zulfiker
authors.affiliation: School of Medical Science, Menzies Health Institute Queensland, Griffith University, Gold Coast 4215, Australia.
authors.forename: Adnan
authors.initials: A
authors.lastname: Ahmetagic
authors.affiliation: School of Medical Science, Menzies Health Institute Queensland, Griffith University, Gold Coast 4215, Australia; School of Physiotherapy, Australian Catholic University, Banyo 4014, Australia.
authors.forename: David
authors.initials: D
authors.lastname: Good
authors.affiliation: School of Medical Science, Menzies Health Institute Queensland, Griffith University, Gold Coast 4215, Australia. Electronic address: m.wei@griffith.edu.au.
authors.forename: Ming Q
authors.initials: MQ
authors.lastname: Wei
mesh_terms: D001419:Bacteria / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D001431:Bacteriological Techniques / Q000379:methods; D004247:DNA / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D059018:DNA Contamination; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D000068536:Bacillota / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D008297:Male; D064307:Microbiota / Q000187:drug effects / Q000235:genetics; D009055:Mouth / Q000382:microbiology*; D009113:Muramidase / Q000737:chemistry; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D012463:Saliva / Q000382:microbiology; D012697:Serine Endopeptidases / Q000737:chemistry; D020298:U937 Cells; D015040:Zirconium / Q000737:chemistry
chemical_list: D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D004247:DNA; D015040:Zirconium; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D009113:Muramidase; D012697:Serine Endopeptidases; C036813:lysyl endopeptidase
keywords: Bacterial cell lysis; DNA; Firmicutes; GAPDH; Oral rinse sample; Total bacteria