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- Collins, M D, et al.
(författare)
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Characterization of Actinomyces isolates from infected root canals of teeth: description of Actinomyces radicidentis sp. nov.
- 2000
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Ingår i: Journal of clinical microbiology. - 0095-1137. ; 38:9, s. 3399-403
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Tidskriftsartikel (refereegranskat)abstract
- Two strains of a previously undescribed Actinomyces-like bacterium were recovered in pure culture from infected root canals of teeth. Analysis by biochemical testing and polyacrylamide gel electrophoresis of whole-cell proteins indicated that the strains closely resembled each other phenotypically but were distinct from previously described Actinomyces and Arcanobacterium species. Comparative 16S rRNA gene-sequencing studies showed the bacterium to be a hitherto unknown subline within a group of Actinomyces species which includes Actinomyces bovis, the type species of the genus. Based on phylogenetic and phenotypic evidence, we propose that the unknown bacterium isolated from human clinical specimens be classified as Actinomyces radicidentis sp. nov. The type strain of Actinomyces radicidentis is CCUG 36733.
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| 6. |
- Johansson, Anders, 1966-
(författare)
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Comparative analysis of PCR versus culture for diagnosis of ulceroglandular tularemia
- 2000
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Ingår i: Journal of Clinical Microbiology. - Washington D.C. : ASM. - 0095-1137 .- 1098-660X. ; 38:1, s. 22-26
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Tidskriftsartikel (refereegranskat)abstract
- PCR and culture were comparatively evaluated for their abilities to demonstrate Francisella tularensis in wound specimens from tularemia patients during an outbreak in Sweden in 1998. For transport of the specimens used for PCR, a buffer solution containing a nuclease inhibitor was used, and for transport of the specimens used for culture, a commercial transport system was selected after experimental comparison of various systems. Of 40 patients with culture- and/or serology-verified ulceroglandular tularemia, PCR detected F. tularensis DNA in 30 (75%) patients, whereas culture detected bacterial growth in 25 (62%) patients. Compared to data from a previous study, the present inclusion of a nuclease inhibitor in the transport medium did not improve the sensitivity of the PCR, whereas the sensitivity of the culture procedure was significantly increased by selection of the system used for transport. Among eight patients with clinically suspected tularemia but with negative serology and culture, specimens from four patients showed detectable DNA. In three of these patients the diagnosis was verified by the demonstration of an F. tularensis-specific T-cell response in vitro. In conclusion, PCR was more sensitive than culture for demonstration of F. tularensis in wound specimens. Besides, we showed that tularemia may proceed without development of serum antibodies, and in these patients, PCR may be of special importance for verification of the diagnosis.
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| 7. |
- Johansson, Anders, 1966-
(författare)
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Evaluation of PCR-based methods for discrimination of Francisella species and subspecies and development of a specific PCR that distinguishes the two major subspecies of Francisella tularensis.
- 2000
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Ingår i: Journal of Clinical Microbiology. - Washington D.C. : ASM. - 0095-1137 .- 1098-660X. ; 38:11, s. 4180-4185
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Tidskriftsartikel (refereegranskat)abstract
- Previous studies have demonstrated that the four subspecies of the human pathogen Francisella tularensis, despite showing marked variations in their virulence for mammals and originating from different regions in the Northern Hemisphere, display a very close phylogenetic relationship. This property has hampered the development of generally applicable typing methods. To overcome this problem, we evaluated the use of PCR for discrimination of the subspecies using various forms of long arbitrary primers or primers specific for repetitive extragenic palindromic sequences (REP) or enterobacterial repetitive intragenic consensus (ERIC) sequences. Patterns generated by use of REP, ERIC, or long arbitrary primers allowed differentiation at the species level and of the four subspecies of F. tularensis. With each of these three methods, similar or identical clustering of strains was found, and groups of strains of different geographical origins or differing in virulence showed distinct patterns. The discriminatory indices of the methods varied from 0.57 to 0.65; thus, the patterns were not sufficiently discriminatory to distinguish individual strains. The sequence of a fragment generated by amplification with an arbitrary primer was determined, and a region showing interstrain heterogeneity was identified. Specific primers were designed, and a PCR was developed that distinguished strains of F. tularensis subsp. holarctica from strains of other F. tularensis subspecies, including strains of the highly virulent F. tularensis subsp. tularensis. Notably, one European isolate showed the genetic pattern typical of the highly virulent F. tularensis subsp. tularensis, generally believed to exist only in North America. It is proposed that a combination of the specific PCR together with one method generating subspecies-specific patterns is suitable as a rapid and relatively simple strategy for discrimination of Francisella species and subspecies.
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- Nelson, J H, et al.
(författare)
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A novel and rapid PCR-based method for genotyping human papillomaviruses in clinical samples.
- 2000
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 38:2, s. 688-95
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Tidskriftsartikel (refereegranskat)abstract
- Many human papillomavirus (HPV) genotypes are associated with cervical carcinoma. We demonstrate the utility of an innovative technique for genotyping of HPV in cervical tissue samples. This method provides an accurate means of identification of the specific HPV genotypes present in clinical specimens. By using the MY09-MY11 and the GP5(+)-GP6(+) consensus primer pairs, HPV sequences were amplified by nested PCR from DNA isolated from cervical smear samples. This led to the production of an approximately 140-bp PCR product from the L1 (major capsid) gene of any of the HPVs present in the sample. PCR was performed with a deoxynucleoside triphosphate mixture which resulted in the incorporation of deoxyuridine into the amplified DNA product at positions where deoxythymidine would normally be incorporated at a frequency of about once or twice per strand. Following the PCR, the product was treated with an enzyme mix that contains uracil N-glycosylase (UNG) and endonuclease IV. UNG removes the uracil base from the nucleotide, and endonuclease IV cleaves the phosphodiester bond at this newly formed abasic site, producing fragments of various sizes. By having end labeled one of the amplification primers, a DNA ladder which is analogous to a "T-sequencing ladder" was produced upon electrophoresis of the products. By comparing this T-sequencing ladder to the known sequences of HPVs, the genotypes of unknown HPV isolates in samples were assigned. Data showing the utility of this technique for the rapid analysis of clinical samples are presented.
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- Nygren, M., et al.
(författare)
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Polymorphism in the pertussis toxin promoter region affecting the DNA-based diagnosis of Bordetella infection
- 2000
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 38:1, s. 55-60
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Tidskriftsartikel (refereegranskat)abstract
- The pertussis toxin (PT) promoter region is a frequently used target for DNA-based diagnosis of pertussis and perapertussis infections. The reported polymorphism in this region has also allowed discrimination of species in mixtures with several Bordetella species by their specific PCR amplicon restriction patterns. In the present study, we investigated the degree of polymorphism in order to confirm the reliability of the assay, Five different sequence types of the amplified 239- or 249-bp region were found among the 33 Bordetella pertussis, B. parapertussis, and B. bronchiseptica American Type Culture Collection reference strains and patient isolates analyzed. According to the sequences that were obtained and according to the PT promoter sequences already available in the databases, restriction enzyme analysis with TaqI, Bg/I, and HaeII, which gave four different patterns, can be performed to reliably identify B. pertussis, B. papapeptussis, and B. bronchiseptica.
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