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dc.contributor.authorMauchline, Tim H.
dc.contributor.authorMohan, Sharad
dc.contributor.authorSchaff, J. E.
dc.contributor.authorOpperman, Charles H.
dc.contributor.authorKerry, Brian R.
dc.contributor.authorDavies, Keith
dc.contributor.authorHirsch, Penny R.
dc.date.accessioned2012-03-20T12:00:39Z
dc.date.available2012-03-20T12:00:39Z
dc.date.issued2010-05
dc.identifier.citationMauchline , T H , Mohan , S , Schaff , J E , Opperman , C H , Kerry , B R , Davies , K & 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 , vol. 50 , no. 5 , pp. 515-521 . https://doi.org/10.1111/j.1472-765X.2010.02830.x
dc.identifier.issn0266-8254
dc.identifier.otherPURE: 589534
dc.identifier.otherPURE UUID: 47bd298f-6ad9-444a-9258-bc91653baf8b
dc.identifier.otherWOS: 000276406400012
dc.identifier.otherScopus: 77950478335
dc.identifier.otherORCID: /0000-0001-6060-2394/work/32215789
dc.identifier.urihttp://hdl.handle.net/2299/7990
dc.description.abstractAims: 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.en
dc.format.extent7
dc.language.isoeng
dc.relation.ispartofLetters in Applied Microbiology
dc.subject16S rRNA gene
dc.subjectbead-beating
dc.subjectendospores
dc.subjectgyrB
dc.subjectmicroLYSIS (R)-PLUS
dc.subjectmultiple strand amplification
dc.subjectPasteuria penetrans
dc.subjectphi29
dc.subjectROOT-KNOT NEMATODES
dc.subjectPLANT-PARASITIC NEMATODES
dc.subjectHYPERPARASITIC BACTERIUM
dc.subjectOBLIGATE ENDOPARASITE
dc.subjectPHYLOGENETIC POSITION
dc.subjectMELOIDOGYNE-INCOGNITA
dc.subjectBIOLOGICAL-CONTROL
dc.subjectSP-NOV
dc.subjectPRATYLENCHUS
dc.subjectHETERODERA
dc.titleA method for release and multiple strand amplification of small quantities of DNA from endospores of the fastidious bacterium Pasteuria penetransen
dc.contributor.institutionDepartment of Human and Environmental Sciences
dc.contributor.institutionHealth & Human Sciences Research Institute
dc.description.statusPeer reviewed
rioxxterms.versionofrecordhttps://doi.org/10.1111/j.1472-765X.2010.02830.x
rioxxterms.typeJournal Article/Review
herts.preservation.rarelyaccessedtrue


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