Real-time detection of Brucella abortus, Brucella melitensis and Brucella suis

Redkar, Rajendra J.; Rose, Sharon; Bricker, Betsy J.; DelVecchio, Vito G.

doi:10.1006/mcpr.2000.0338

Cited by 118 publications

(84 citation statements)

References 23 publications

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“…There were some previous trials to discriminate among the species of the genus Brucella using the wide possibilities of PCR techniques (Fekete et al, 1992b;Bricker andHalling, 1994,1995;DaCosta et al, 1996;Ouahrani-Bettach et al, 1996;Techerneva et al, 1996Techerneva et al, , 2000Sifuentes et al, 1997;Redkar et al, 2001;Ocampo-Sosa, 2005;Ferrao-Beck et al, 2006;GarciaYoldi et al, 2006), who obtained similar results to that obtained in the present study.…”

Section: Resultssupporting

confidence: 87%

“…As it is much easier and more valuable to identify an isolate as S19, RB51 or any other Brucella strain, the assay was modified (Bricker and Halling, 1995), by the introduction of new primers (Eri1, Eri2 and RB51/2308 primer) that can detect and differentiate B. abortus S19 vaccine strain and B. abortus strain RB51 and/ or its parent strain 2308. The test was previously evaluated and successfully applied as a diagnostic tool (Fekete et al, 1992; Leal-Klevezas et al, 1995; Romero et al, 1995a;1995b;Ewalt and Bricker, 2000;Adon et al, 2001), applied with modified version (Redkar et al, 2001;Ewalt and Bricker, 2003;Ocampo-Sosa et al, 2005). Amplification of both 731 and 178 bp indicates that the strain is B. melitensis.…”

Section: Resultsmentioning

confidence: 99%

“…The differential identification of Brucella species (differential PCR-based assay), that depends on strain locus specific multiplexing (e.g. AMOS-PCR based on IS711, PCR-RFLP or RAPD-PCR), (Bricker and Halling, 1994;Bricker and Halling, 1995;Sifuentes et al, 1997;Tcherneva et al, 2000;Adone et al, 2001;Redkar et al, 2001;Probert et al, 2004;Ocampo-Sosa et al, 2005). Differential PCR based assays are particularly useful for epidemiological trace back or for species-specific eradication programs (Bricker, 2002).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Evaluation of the currently used polymerase chain reaction assays for molecular detection of Brucella species

Moussa¹,

Omnia²,

Amin³

et al. 2011

Afr. J. Microbiol. Res.

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The diagnosis of brucellosis is the corner stone in any control and eradication program. Therefore, the main objective of the present study was to apply more advanced techniques for rapid and accurate diagnosis of brucellosis that can overcome the draw backs of the traditional diagnostic techniques. Different polymerase chain reaction (PCR) assays were applied in the present study, either singly or in a multiplex format, that enable to detect and differentiate most of Brucella species. The PCR assay detection limit was evaluated in a preliminary study. The obtained results recommend the PCR assay as a valuable, rapid, very specific, highly sensitive and safe laboratory diagnostic test that can be used not only for detection of Brucella antigen either in culture or in clinical samples but also in differentiating most of the virulent and vaccinal strains.

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Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Evaluation of the currently used polymerase chain reaction assays for molecular detection of Brucella species

Moussa¹,

Omnia²,

Amin³

et al. 2011

Afr. J. Microbiol. Res.

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“…The per gene is, with various degrees of similarity, present in a few other bacteria, including Yersinia enterocolitica serotype O:9, Vibrio cholerae O1, Escherichia coli O:157, some serovars of E. hermanni and Stenotrophomonas maltophilia, and Salmonella group N (O:30) (14). To the best of our knowledge, the only real-time PCR work reported is based on hybridization probes used in three different assays for identification of three Brucella species (15). This report describes for the first time the development of a ready-to-go, nonproprietary, open-formula (thus possessing the potential for standardization), 5Ј hydrolysis probe-using real-time PCR assay including an internal amplification control (IAC) for direct verification of suspected Brucella colonies on agar plates.…”

mentioning

confidence: 99%

Validated 5′ Nuclease PCR Assay for Rapid Identification of the Genus Brucella

et al. 2004

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A real-time, genus-specific 5 nuclease PCR assay for amplification of a 322-bp fragment of the per gene was developed for rapid (<2 h) identification of Brucella spp. from agar plates. The assay, including an internal amplification control (116 bp), identified Brucella strains (n ‫؍‬ 23) and did not detect non-Brucella strains (n ‫؍‬ 174), indicating its usefulness, particularly for laboratories with stringent quality assurance programs.Owing to continuous efforts to control and eradicate brucellosis in domestic animals, the levels of brucellosis have been reduced in many countries (6). However, a natural reservoir of Brucella bacteria in wildlife, e.g., Brucella suis biovar 2 in hares in Denmark, can still pose a threat. Thus, the task of detection and identification remains challenging and requires reliable and sensitive diagnostics tools.The diagnosis of brucellosis is based mainly on serological responses, which can be unspecific owing to cross-reaction or subsensitive reactions in samples from areas with a low or subclinical prevalence of brucellosis (5,7,9,16). Culture-based verification of suspected cases, or pathological findings in clinical cases, can be time-consuming and also can impose a hazard to laboratory personal. Thus, numerous alternative verification methods, based mostly on amplification of universal genes in a conventional PCR, have been reported, although some have produced false-positive results (reviewed in reference 3).The Brucella-specific perosamine synthetase (per) gene is highly conserved and present even in the naturally rough Brucella species B. ovis and B. canis and spontaneously rough strains of B. abortus and B. melitensis (4). The per gene is, with various degrees of similarity, present in a few other bacteria, including Yersinia enterocolitica serotype O:9, Vibrio cholerae O1, Escherichia coli O:157, some serovars of E. hermanni and Stenotrophomonas maltophilia, and Salmonella group N (O:30) (14). To the best of our knowledge, the only real-time PCR work reported is based on hybridization probes used in three different assays for identification of three Brucella species (15). This report describes for the first time the development of a ready-to-go, nonproprietary, open-formula (thus possessing the potential for standardization), 5Ј hydrolysis probe-using real-time PCR assay including an internal amplification control (IAC) for direct verification of suspected Brucella colonies on agar plates.The Brucella-specific primers were designed as previously described (11). The primer combination bruc1-bruc5 was found to be most selective (11). In the present study, three different TaqMan probes (6-carboxyfluorescein [FAM] labeled) were designed and compared in the assay: Bruc1, in close proximity to the 3Ј end of the forward primer; Bruc2, in the middle of the amplified fragment; and Bruc3, within a few nucleotides of the 3Ј end of the reverse primer (Table 1).An artificially created chimerical DNA (12), a second set of primers, and a second TaqMan probe (VIC labeled) (12) were used f...

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“…The advantage of this method is due to the strict spatial limitation for observing FRET. In this case, non-bound probes in solution exhibit only a minimal background so that lower detection limits can be achieved [1,[3][4][5].…”

Section: Introductionmentioning

confidence: 99%

Real-time quantification of RNA polymerase activity using a “broken beacon”

Blair

Rosenblum

Dawson

et al. 2007

Analytical Biochemistry

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A novel assay was developed for real-time monitoring of RNA synthesis using a hybridization-based method. In this work, a "broken beacon" in which the fluor and quencher were located on separate but complementary oligonucleotides was used to quantify the amount of RNA production by T7 polymerase. The relative lengths of the fluor-oligo and quencher-oligo, as well as their relative concentrations, were optimized. The experimentally determined limit-of-detection was ~45 nM. The new assay was compared to the "gold-standard" radiolabel ([ 32 P]NTP incorporation) assay for RNA quantification. While the broken beacon assay exhibited a higher limit of detection, it provided an accurate measure of RNA production rates. However, the broken beacon assay provided the significant analytical advantages of i) a real-time and continuous measurement, ii) no requirement for the use of radiolabels or gel-based analysis, and iii) substantial time and labor savings.

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Real-time detection of Brucella abortus, Brucella melitensis and Brucella suis

Cited by 118 publications

References 23 publications

Evaluation of the currently used polymerase chain reaction assays for molecular detection of Brucella species

Evaluation of the currently used polymerase chain reaction assays for molecular detection of Brucella species

Validated 5′ Nuclease PCR Assay for Rapid Identification of the Genus Brucella

Real-time quantification of RNA polymerase activity using a “broken beacon”

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