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Updated: Jun 14, 2026
Physical Isolation of Endospores from Environmental Samples by Targeted Lysis of Vegetative Cells
Published on: January 21, 2016
A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious
T H Mauchline1, S Mohan, K G Davies
1Nematode Interactions Unit, Department of Plant Pathology and Microbiology, Rothamsted Research, Harpenden, Hertfordshire, UK. tim.mauchline@bbsrc.ac.uk
Aims:
To establish a reliable protocol to extract DNA from Pasteuria penetrans endospores for use as template in multiple strand amplification, thus providing sufficient material for genetic analyses. To develop a highly sensitive PCR-based diagnostic tool for P. penetrans.
Methods And Results:
An optimized method to decontaminate endospores, release and purify DNA enabled multiple strand amplification. DNA purity was assessed by cloning and sequencing gyrB and 16S rRNA gene fragments obtained from PCR using generic primers. Samples indicated to be 100%P. penetrans by the gyrB assay were estimated at 46% using the 16S rRNA gene. No bias was detected on cloning and sequencing 12 housekeeping and sporulation gene fragments from amplified DNA. The detection limit by PCR with Pasteuria-specific 16S rRNA gene primers following multiple strand amplification of DNA extracted using the method was a single endospore.
Conclusions:
Generation of large quantities DNA will facilitate genomic sequencing of P. penetrans. Apparent differences in sample purity are explained by variations in 16S rRNA gene copy number in Eubacteria leading to exaggerated estimations of sample contamination. Detection of single endospores will facilitate investigations of P. penetrans molecular ecology.
Significance And Impact Of The Study:
These methods will advance studies on P. penetrans and facilitate research on other obligate and fastidious micro-organisms where it is currently impractical to obtain DNA in sufficient quantity and quality.
Insights
A new DNA extraction protocol for Pasteuria penetrans endospores allows for multiple strand amplification and sensitive PCR detection. This method can detect single endospores, advancing molecular ecology studies.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Pasteuria penetrans is an obligate endospore-forming bacterium.
- Obtaining sufficient DNA from P. penetrans endospores for genetic analysis is challenging.
- A sensitive diagnostic tool for P. penetrans is needed.
Purpose of the Study:
- To develop a reliable protocol for DNA extraction from P. penetrans endospores.
- To enable multiple strand amplification for genetic analyses.
- To create a highly sensitive PCR-based diagnostic tool for P. penetrans.
Main Methods:
- Optimized decontamination, DNA release, and purification of endospores.
- Multiple strand amplification of extracted DNA.
- PCR using generic and Pasteuria-specific primers for gene fragment analysis (gyrB, 16S rRNA).
Main Results:
- The optimized method enabled multiple strand amplification and DNA purification.
- DNA purity was assessed via cloning and sequencing of gene fragments.
- PCR with Pasteuria-specific primers achieved a detection limit of a single endospore.
Conclusions:
- The protocol facilitates genomic sequencing of P. penetrans.
- Variations in 16S rRNA gene copy number explain apparent differences in sample purity.
- Detection of single endospores aids P. penetrans molecular ecology research and studies of other fastidious microorganisms.
