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High-Quality Nucleic Acid Isolation from Hard-to-Lyse Bacterial Strains Using PMAP-36, a Broad-Spectrum Antimicrobial Peptide
- Source :
- International Journal of Molecular Sciences, International Journal of Molecular Sciences, Vol 22, Iss 4149, p 4149 (2021), Volume 22, Issue 8
- Publication Year :
- 2021
- Publisher :
- MDPI, 2021.
-
Abstract
- The efficiency of existing cell lysis methods to isolate nucleic acids from diverse bacteria varies depending on cell wall structures. This study tested a novel idea of using broad-spectrum antimicrobial peptides to improve the lytic efficiency of hard-to-lyse bacteria and characterized their differences. The lysis conditions of Staphylococcus aureus using recombinant porcine myeloid antimicrobial peptide 36 (PMAP-36), a broad-spectrum pig cathelicidin, was optimized, and RNA isolation was performed with cultured pellets of ten bacterial species using various membranolytic proteins. Additionally, three other antimicrobial peptides, protegrin-1 (PG-1), melittin, and nisin, were evaluated for their suitability as the membranolytic agents of bacteria. However, PMAP-36 use resulted in the most successful outcomes in RNA isolation from diverse bacterial species. The amount of total RNA obtained using PMAP-36 increased by ~2-fold compared to lysozyme in Salmonella typhimurium. Streptococci species were refractory to all lytic proteins tested, although the RNA yield from PMAP-36 treatment was slightly higher than that from other methods. PMAP-36 use produced high-quality RNA, and reverse transcription PCR showed the efficient amplification of the 16S rRNA gene from all tested strains. Additionally, the results of genomic DNA isolation were similar to those of RNA isolation. Thus, our findings present an additional option for high quality and unbiased nucleic acid isolation from microbiomes or challenging bacterial strains.
- Subjects :
- 0301 basic medicine
DNA, Bacterial
Staphylococcus aureus
Lysis
QH301-705.5
Antimicrobial peptides
Cell Fractionation
Catalysis
Article
Microbiology
Inorganic Chemistry
03 medical and health sciences
antimicrobial peptides
0302 clinical medicine
PMAP-36
Physical and Theoretical Chemistry
Biology (General)
Molecular Biology
QD1-999
Spectroscopy
nucleic acids isolation
biology
Chemistry
Organic Chemistry
RNA
General Medicine
Antimicrobial
biology.organism_classification
Computer Science Applications
genomic DNA
RNA, Bacterial
030104 developmental biology
030220 oncology & carcinogenesis
Nucleic acid
RNA extraction
Bacteria
Antimicrobial Cationic Peptides
Subjects
Details
- Language :
- English
- ISSN :
- 14220067
- Volume :
- 22
- Issue :
- 8
- Database :
- OpenAIRE
- Journal :
- International Journal of Molecular Sciences
- Accession number :
- edsair.doi.dedup.....6560557911fd8b2d68aba608cd5b56be