Taleski, Vaso (2009) “PCR based methods for diagnosis of human brucellosis". In: Microbiologia Balkanica 2009 & 4-ti Kongres na mikrobiolozite na Makedonija, 28-31 October 2009, Ohrid, Macedonia.
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Abstract
Introduction
Human brucellosis has been attributed to B. abortus, B. melitensis, B. suis, and B. canis and more recently to strains resembling Brucella isolated from marine mammals. Brucella has been isolated from human tissue samples, blood, urine, cerebrospinal fluid which are suitable for analysis by PCR.
Brucella species are highly monomorphic with minimal genetic variation among species.
Brucella genome consists of two circular hromosomes, has been completely sequenced for B. melitensis, B.abortus and B. suis. B. melitensis genome contains 3,294,931 base pairs (bp) - chromosome I of 2,117,144 bp and chromosome II of 1,177,787 bp. Brucella abortus chromosome I contains 2,124,241 bp and chromosomes II is 1,162,204 bp.
Methods
Genes encoding for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis can be found on both chromosomes.
The first PCR based assays, used for genus-specific identification of Brucella, amplified genes encoding: 43-kDa outer membrane protein of B. abortus (primers NP, amplicon size 635 bp); 31-kDa B. abortus protein (primers B4/B5, size 223 bp); omp-2/ membrane extern B. abortus protein (JPF/JPR, size 193 bp); B. abortus 16S rRNA (Ba148-167F/Ba928-948R primers, size 800 bp); B. abortus 16S rRNA (F4/R2 primers, size 905 bp).
The most frequently described PCR target for the diagnosis of human brucellosis is the bcsp31 gene encoding a 31-kDa antigen conserved among Brucella spp.
An insertion sequence (IS711) element named IS6501, 836 bp in length, occurs 20-35 times in the B. ovis genome and 5-15 times in other Brucella species. Most Brucella species contain at least one copy of IS711 at a unique chromosomal location. The multiplex assay consists of one common primer anchored in the IS711 and a species-specific primer that binds to the unique sequence allowed species identification determined by the size of the amplicon.
Results
In our research with R.A.P.I.D. PCR detecting Brucella DNA from peripheral blood samples, using specific set of primers for IS711 and bcsp31 showed: sensitivity of 56% (bcsp31), 10% (IS711) and specificity of 100%.
Conclusion
The advantages of PCR for diagnosis of human brucellosis are its high specificity and sensitivity, rapid detection and identification. Two possible limitations of PCR are: false-positive as a result of contamination and false negative due presence of inhibitory compounds. Effective PCR detection of Brucella from peripheral blood in order to get a better results requires sampling at the beginning of the disease, more efficient concentration techniques or larger volumes of blood for processing.
| Item Type: | Conference or Workshop Item (Lecture) |
|---|---|
| Subjects: | Medical and Health Sciences > Basic medicine Medical and Health Sciences > Health sciences |
| Divisions: | Faculty of Medical Science |
| Depositing User: | Vaso Taleski |
| Date Deposited: | 26 Dec 2012 15:17 |
| Last Modified: | 26 Dec 2012 15:17 |
| URI: | https://eprints.ugd.edu.mk/id/eprint/4578 |
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