Please use this identifier to cite or link to this item: http://ahro.austin.org.au/austinjspui/handle/1/9948
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dc.contributor.authorGürtler, Volkeren
dc.contributor.authorHarford, Cateen
dc.contributor.authorBywater, Judyen
dc.contributor.authorMayall, Barrie Cen
dc.date.accessioned2015-05-15T23:14:29Z
dc.date.available2015-05-15T23:14:29Z
dc.date.issued2005-06-24en
dc.identifier.citationJournal of Microbiological Methods 2005; 64(2): 185-99en
dc.identifier.govdoc15979743en
dc.identifier.otherPUBMEDen
dc.identifier.urihttp://ahro.austin.org.au/austinjspui/handle/1/9948en
dc.description.abstractTo obtain Mycobacterium species identification directly from clinical specimens and cultures, the 16S-23S rDNA spacer (ISR) was amplified using previously published primers that detect all Mycobacterium species. The restriction enzyme that could potentially produce the most restriction fragment length polymorphisms (RFLPs) was determined from all available ISR DNA sequences in GenBank to produce a novel data set of RFLPs for 31 slowly growing Mycobacterium species. Subsequently a GelCompar II database was constructed from RFLPs for 10 enzymes that have been used in the literature to differentiate slowly growing Mycobacterium species. The combination of Sau96I and HaeIII were the best choice of enzymes for differentiating clinically relevant slowly growing Mycobacterium species. A total of 392 specimens were studied by PCR with 195 negative and 197 positive specimens. The ISR-PCR product was digested with HaeIII (previously reported) and Sau96I (new to this study) to obtain a Mycobacterium species identification based on the ISR-RFLPs. The species identification obtained by ISR-RFLP was confirmed by DNA sequencing (isolate numbers are shown in parentheses) for M. avium (3), M. intracellulare (4), M. avium complex (1), M. gordonae (2) and M. tuberculosis (1). The total number of specimens (99) identified were from culture (67), Bactectrade mark 12B culture bottles (11), EDTA blood (3), directly from smear positive specimens (13), tissue (4) and urine (1). Direct species identification was obtained from all 13/13 smear positive specimens. The total number of specimens (99) were identified as M. tuberculosis (41), M. avium (7), M. avium complex (11), M. intracellulare MIN-A (20), M. flavescens (2), M. fortuitum (10), M. gordonae (4), M. shimoidei (1), M. ulcerans (1) and M. chelonae (2). This method reduces the time taken for Mycobacterium species identification from 8-10 weeks for culture and biochemical identification; to 4-6 weeks for culture and ISR-RFLP; to 2 days for smear-positive specimens by ISR-RFLP. The precise 2 day identification obtained may provide significant advantages in clinical management.en
dc.language.isoenen
dc.subject.otherBacterial Typing Techniques.methodsen
dc.subject.otherBase Sequenceen
dc.subject.otherDNA, Bacterial.geneticsen
dc.subject.otherDNA, Ribosomal Spacer.geneticsen
dc.subject.otherDeoxyribonucleases, Type II Site-Specificen
dc.subject.otherHumansen
dc.subject.otherMolecular Sequence Dataen
dc.subject.otherMycobacterium.classification.genetics.growth & development.isolation & purificationen
dc.subject.otherMycobacterium Infections.diagnosis.microbiologyen
dc.subject.otherPolymerase Chain Reactionen
dc.subject.otherPolymorphism, Restriction Fragment Lengthen
dc.subject.otherRNA, Bacterial.geneticsen
dc.subject.otherRNA, Ribosomal, 16S.geneticsen
dc.subject.otherRNA, Ribosomal, 23S.geneticsen
dc.subject.otherSpecies Specificityen
dc.titleDirect identification of slowly growing Mycobacterium species by analysis of the intergenic 16S-23S rDNA spacer region (ISR) using a GelCompar II database containing sequence based optimization for restriction fragment site polymorphisms (RFLPs) for 12 enzymes.en
dc.typeJournal Articleen
dc.identifier.journaltitleJournal of microbiological methodsen
dc.identifier.affiliationVolker.Gurtler@austin.org.auen
dc.identifier.affiliationDepartment of Microbiology, Austin Health, Studley Road, Heidelberg, Victoria 3084, Australiaen
dc.identifier.doi10.1016/j.mimet.2005.04.034en
dc.description.pages185-99en
dc.relation.urlhttp://www.ncbi.nlm.nih.gov/pubmed/15979743en
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