Efficiency of chemical versus mechanical disruption methods of DNA extraction for the identification of oral Gram-positive and Gram-negative bacteria

• Published in: Journal of international medical research Vol. 48; no. 5; p. 300060520925594
• Main Authors: Li, Xiaolan, Bosch-Tijhof, Caroline J., Wei, Xi, de Soet, Johannes J., Crielaard, Wim, Loveren, Cor van, Deng, Dong Mei
• Format: Journal Article
• Language: English
• Published: London, England SAGE Publications 01-05-2020; Sage Publications Ltd; SAGE Publishing
• Subjects: Automation; Bacteria; Bacterial Typing Techniques - instrumentation; Bacterial Typing Techniques - methods; Deoxyribonucleic acid; DNA; DNA, Bacterial - genetics; DNA, Bacterial - isolation & purification; Efficiency; Genome, Bacterial - genetics; Gram-Negative Bacteria - genetics; Gram-Negative Bacteria - isolation & purification; Gram-positive bacteria; Gram-Positive Bacteria - genetics; Gram-Positive Bacteria - isolation & purification; Humans; Methods; Pre-Clinical Research Report; Real-Time Polymerase Chain Reaction; Saliva - microbiology; Specimen Handling - methods; saliva; molecular diagnostics; Genomic DNA; in vitro; Gram-positive bacteria; Gram-negative bacteria; extraction methods
• ISSN: 0300-0605; 1473-2300
• DOI: 10.1177/0300060520925594

Objective
Clinical diagnostics often requires the detection of multiple bacterial species in limited clinical samples with a single DNA extraction method. This study aimed to compare the bacterial DNA extraction efficiency of two lysis methods automated with the MagNA-Pure LC instrument. The samples included five oral bacterial species (three Gram-positive and two Gram-negative) with or without human saliva background.
Methods
Genomic DNA (gDNA) was extracted from bacterial cultures by bead-beating lysis (BMP) or chemical lysis (MP), followed by automated purification and measurement by quantitative PCR.
Results
For pure bacterial cultures, the MP method yielded higher quantities of extracted DNA and a lower detection limit than the BMP method, except where the samples contained high numbers of Gram-positive bacteria. For bacterial cultures with a saliva background, no difference in gDNA extraction efficacy was observed between the two methods.
Conclusions
The efficiency of a bacterial DNA extraction method is not only affected by the bacterial cell wall structure but also by the sample milieu. The MP method provided superior gDNA extraction efficiency when the samples contained a single bacterial species, whereas either of the BMP and MP methods could be applied with similar efficiencies to samples containing multiple species of bacteria.