A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans

A - Papers appearing in refereed journals

Mauchline, T. H., Mohan, S., Davies, K. G., Schaff, J. E., Opperman, C. H., Kerry, B. R. and Hirsch, P. R. 2010. A method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetrans. Letters in Applied Microbiology. 50 (5), pp. 515-521.

AuthorsMauchline, T. H., Mohan, S., Davies, K. G., Schaff, J. E., Opperman, C. H., Kerry, B. R. and Hirsch, P. R.
Abstract

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.

KeywordsBiotechnology & Applied Microbiology; Microbiology
Year of Publication2010
JournalLetters in Applied Microbiology
Journal citation50 (5), pp. 515-521
Digital Object Identifier (DOI)doi:10.1111/j.1472-765X.2010.02830.x
PubMed ID20302597
Open accessPublished as non-open access
FunderBiotechnology and Biological Sciences Research Council
British Council through the United Kingdom-India Education and Research Initiative (UKIERI)
Funder project or codeSEF
ISSN02668254
PublisherWiley

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