Spore-DNA localization and extraction efficiencies of Bacillus subtilis for accurate results in quantitative real-time polymerase chain reaction.
J Microorg Control
; 29(1): 9-15, 2024.
Article
in En
| MEDLINE
| ID: mdl-38508764
ABSTRACT
Mechanical bead disruption is an efficient DNA extraction method from spore cells for subsequent quantification of the spore population by quantitative polymerase chain reactionï¼qPCRï¼. In this study, to validate spore DNA localization and extraction efficiencies, the fractionated DNA included the total DNAï¼tDNAï¼extracted from spore cells and intracellularï¼iDNAï¼and extracellular DNAï¼eDNAï¼extracted from fractionated spores through chemical decoating and alkaline lysis buffers, each followed by bead disruption. Furthermore, alkaline lysis buffer-treated spore cells were intensively washed three and five times after each centrifugation to determine how the amount of DNA is affected by repeated centrifugation. This process was achieved through fractionated spore pellet and suspension treatments with propidium monoazide xxï¼PMAxxï¼before mechanical bead disruption. Three fractionated and extracted DNAs were assessed with qPCR. The amount of eDNA was higher than that of iDNA, and closer to tDNA levels in the qPCR assay. These results indicted the following 1ï¼amount of eDNA was more than iDNA and responsible for majority of amount of tDNA through the combination method involving alkaline lysis buffer and bead disruption, 2ï¼lysis buffer partially eliminated the eDNA fragments through multiple washing steps, but it was not largely independent of the number of times centrifugation was performed.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Spores, Bacterial
/
Bacillus subtilis
Language:
En
Journal:
J Microorg Control
Year:
2024
Document type:
Article