Novel identification and differentiation of Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae suitable for both conventional and real-time PCR systems

Hinić, Višnja; Brodard, Isabelle; Thomann, Andreas; Cvetnić, Željko; Makaya, Pious V.; Frey, Joachim; Abril, Carlos

doi:10.1016/j.mimet.2008.07.002

Cited by 134 publications

(96 citation statements)

References 21 publications

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“…Current microbiological (4) and low-resolution molecular typing methods are useful for identifying Brucella isolates and determining species and biovar designations; however, they have limited value for epidemiological trace-back investigations (6,15). High-resolution genetic subtyping tools that could provide important information about disease transmission patterns and the molecular epidemiology of Brucella have been difficult to develop and implement due to the genetically monomorphic nature of Brucella species (12).…”

mentioning

confidence: 99%

Comparison of Two Multiple-Locus Variable-Number Tandem-Repeat Analysis Methods for Molecular Strain Typing of Human Brucella melitensis Isolates from the Middle East

Tiller

Boshra

et al. 2009

J Clin Microbiol

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Brucella species are highly monomorphic, with minimal genetic variation among species, hindering the development of reliable subtyping tools for epidemiologic and phylogenetic analyses. Our objective was to compare two distinct multiple-locus variable-number tandem-repeat analysis (MLVA) subtyping methods on a collection of 101 Brucella melitensis isolates from sporadic human cases of brucellosis in Egypt (n ‫؍‬ 83), Qatar (n ‫؍‬ 17), and Libya (n ‫؍‬ 1). A gel-based MLVA technique, MLVA-15 IGM , was compared to an automated capillary electrophoresis-based method, MLVA-15 NAU , with each MLVA scheme examining a unique set of variable-number tandem repeats. Both the MLVA IGM and MLVA NAU methods were highly discriminatory, resolving 99 and 101 distinct genotypes, respectively, and were able to largely separate genotypes from Egypt and Qatar. The MLVA-15 NAU scheme presented higher strain-to-strain diversity in our test population than that observed with the MLVA-15 IGM assay. Both schemes were able to genetically correlate some strains originating from the same hospital or region within a country. In addition to comparing the genotyping abilities of these two schemes, we also compared the usability, limitations, and advantages of the two MLVA systems and their applications in the epidemiological genotyping of human B. melitensis strains.Brucellosis is a common zoonotic disease causing infections in economically important livestock, such as cattle, goats, sheep, and pigs (22). In humans, this highly diverse illness, also known as Malta or undulant fever, initially presents as a fever, malaise, and myalgia and may later develop into a chronic illness affecting various organs and tissues (21,25). Brucellosis is usually transmitted to humans through consumption of contaminated and untreated milk products or by direct contact with infected animals. There are nine phenotypically recognized species in the Brucella genus: Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae; two new marine species, B. ceti and B. pinnipedialis (11); and B. microti (27), isolated from the common vole. Each species has distinctive host preferences, pathogenicity, and epidemiology.Although some developed countries have eradicated B. abortus from cattle through vaccination campaigns, B. melitensis, B. abortus, and B. suis remain the principal causes of human brucellosis worldwide and are major public health problems, primarily in developing countries (7). Brucellosis is prevalent in the Mediterranean basin, the Middle East, Africa, Asia, and areas of Latin America where people are economically dependent on ruminant livestock (23,28). Over the past decade, the global epidemiology of human brucellosis has changed, due in part to the implementation of national and international surveillance programs, animal vaccination campaigns, and socioeconomic changes (22).Current microbiological (4) and low-resolution molecular typing methods are useful for identifying Brucella isolates and determining species and biovar designatio...

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mentioning

confidence: 99%

Comparison of Two Multiple-Locus Variable-Number Tandem-Repeat Analysis Methods for Molecular Strain Typing of Human Brucella melitensis Isolates from the Middle East

Tiller

Boshra

et al. 2009

J Clin Microbiol

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“…However, sensitivity of the omp2a LAMP assay was equivalent to TaqMan probe based real time PCR. The IS711 TaqMan probe based real time PCR has been reported as a highly sensitive method in detecting less than 10 fg of genomic DNA (three genome copies) of Brucella organisms [17,23,24].…”

Section: Resultsmentioning

confidence: 99%

“…The TaqMan probe based real-time PCR was carried out as reported earlier [17]. In brief, the 25 lL reaction volume consisted of 0.2 lM of probe, 0.4 lM each of primers and 0.5 lL of isolated DNA in 1X probe Mastermix (Sigma, EU).…”

Section: Conventional Pcr and Taqman Probe Based Realtime Pcr Assaysmentioning

confidence: 99%

Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay

et al. 2016

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Several pathogens including Brucella spp. are shed in semen of infected bulls and can be transmitted to cows through contaminated semen during artificial insemination. The present study reports omp2a and bcsp31 gene based loop-mediated isothermal amplification (LAMP) assays for detection of Brucella genomic DNA in semen from infected bulls. The positive results could be interpreted visually by change in colour of reaction mixture containing hydroxyl naphthol blue (HNB) dye from violet to sky blue. LAMP assays based on omp2a and bcsp31 could detect as little as 10 and 100 fg of B. abortus S19 genomic DNA, respectively. Sensitivity of omp2a and bcsp31 LAMP assays for direct detection of organisms in bovine semen was 2.28 9 10 1 CFU and 2.28 9 10 2 CFU of B. abortus S19 in spiked bovine semen, respectively. The omp2a LAMP assay was found equally sensitive to TaqMan probe based real-time PCR and 100 times more sensitive than conventional PCR in identifying Brucella in spiked semen. The diagnostic applicability of the omp2a LAMP assay was evaluated with seventy-nine bovine semen samples and results were re-evaluated through TaqMan probe based real-time PCR and conventional PCR. Taken together, the omp2a LAMP assay is easy to perform, rapid and sensitive in diagnosis of Brucella spp. in bovine semen.

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“…PCR convencional. As amostras de DNA de tecido, homogeneizado e DNA bacteriano foram submetidas à técnica de PCR convencional, utilizando-se como alvo dois genes, sendo eles: IS711 e BruAb2_0168 (Hinić et al 2008), que identificam o gênero e a espécie B. abortus, respectivamente. As sequências dos oligonucleotídeos denominados IS711 e BruAb2_0168 e, o tamanho aproximado dos fragmentos que estes amplificam, encontram-se descritos no Quadro 1.…”

Section: Methodsunclassified

Detecção de Brucella abortus em tecidos bovinos utilizando ensaios de PCR e qPCR¹

Caitano

Soares²,

Ramos³

et al. 2014

Pesq. Vet. Bras.

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The aim of the study was to evaluate the technical polymerase chain reaction (PCR) and Real-Time PCR (qPCR) to detect Brucella abortus from bovine tissues with suggestive lesions of brucellosis. For this, 21 fragments of bovine tissues collected at abattoirs of Mato Grosso do Sul were processed and subjected to microbiological culture and extraction of genomic DNA to perform the PCR reactions and qPCR. Eight samples of microbiological culture showed bacterial growth and five samples were confirmed as B. abortus by PCR. DNA of Brucella (IS711 primers) was detected in 13 (61.9%) directly from tissue samples and 17 (81%) from tissue homogenate samples. With the species-specific set of primers BruAb2_0168F and BruAb2_0168R, 14 (66%) tissue samples and 18 (85.7%) tissue homogenate samples were positive. Six positive samples in the species-specific PCR were sequenced and the best hit in the BLASTn analysis was B. abortus. By qPCR, 21 (100%) tissue samples and 19 (90.5%) tissue homogenate samples were positive for B. abortus. Ten samples of DNA from bovine blood from an accredited-free herd were used as negative control in PCR and qPCR analysis using the primers BruAb2_0168F and BruAb2_0168R, and no one amplified by PCR, whereas two samples were amplified by qPCR (20%). In conclusion, both techniques detect the presence of B. abortus directly from tissues and homogenized, but the qPCR showed high sensitivity. The results indicate that qPCR can represent an alternative tool for faster and more accurate detection of B. abortus directly from tissues, and use in health surveillance programs by presenting satisfactory sensitivity and specificity.

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Novel identification and differentiation of Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae suitable for both conventional and real-time PCR systems

Cited by 134 publications

References 21 publications

Comparison of Two Multiple-Locus Variable-Number Tandem-Repeat Analysis Methods for Molecular Strain Typing of Human Brucella melitensis Isolates from the Middle East

Comparison of Two Multiple-Locus Variable-Number Tandem-Repeat Analysis Methods for Molecular Strain Typing of Human Brucella melitensis Isolates from the Middle East

Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay

Detecção de Brucella abortus em tecidos bovinos utilizando ensaios de PCR e qPCR¹

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