Simultaneous detection of the Genus Brucella by combinatorial PCR

Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.
Japanese journal of infectious diseases (Impact Factor: 1.16). 06/2007; 60(2-3):137-9.
Source: PubMed


We have developed a combinatorial polymerase chain reaction (PCR) procedure to identify four major species of the genus Brucella simultaneously. Four pairs of primers targeting the genes encoding a cell surface protein (BCSP31) and outer membrane proteins (omp2b, omp2a and omp31) were prepared. PCR using these primers gave rise to specific patterns of amplification for each Brucella spp. examined in this study. B. abortus could be identified when fragments of BCSP31 and omp2b/2a were amplified by B. abortus-specific primers. B. melitensis could be identified by the amplification of fragments of BCSP31, omp2b/2a and omp31 using pair of primers B4/B5, JRF/JPR-ab and omp31. Identification of B. canis could be achieved when the amplicons of omp2b/2a were detected by B. canis-specific primers, as could the identification of BCSP31 and omp31. If specific amplifications occurred using all pairs of primers, the strain was identified as B. suis. Combinatorial PCR reported here thus appeared to be an ideal method of identifying Brucella spp., the causative pathogen of human brucellosis.

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    • "For the detection of Brucella species, a multiplex realtime PCR protocol was used, targeting the bcsp31, alkB, and BMEI1162 genes of Brucella species, B. abortus, and B. melitensis, respectively, as previously described [17]. Where it was positive for Brucella species but negative for the other two, B. canis was tested using a PCR protocol targeting the omp2B gene as previously described [18]. For the detection of Leishmania species a real-time PCR protocol targeting the SSU rRNA gene was used, as previously described [19]. "
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    ABSTRACT: The golden jackal Canis aureus occurs in south-eastern Europe, Asia, the Middle East, the Caucasus, and Africa. In Serbia, jackals neared extinction; however, during the last 30 years, the species started to spread quickly and to increase in number. Few studies in the past have revealed their potential role as carriers of zoonotic diseases. Animal samples were collected over a three-year period (01/2010–02/2013) from 12 sites all over Serbia. Of the tissue samples collected, spleen was chosen as the tissue to proceed; all samples were tested for Leishmania species and Brucella species by real-time PCR. Of the 216 samples collected, 15 (6.9%) were positive for Leishmania species, while four (1.9%) were positive for B. canis. The potential epidemiologic role of the golden jackal in carrying and dispersing zoonotic diseases in Serbia should be taken under consideration when applying surveillance monitoring schemes.
    BioMed Research International 05/2014; 2014. DOI:10.1155/2014/728516 · 1.58 Impact Factor
    • "The genome sequences allow construction of general brucella microarrays to observe the dance between microbe and host in understanding the course of brucellosis infection (Halling et al. 2005). Other molecular tools have also been readied, used on the brucellae and published: AFLP (Whatmore et al. 2005), tandemrepeat markers (Whatmore et al. 2006), combinatorial PCR (Imaoka et al. 2007), multilocus sequencing (Whatmore et al. 2007), and so on. The main purpose of these papers is to find a way to identify individual strains so that they can be tracked in detailed epidemiological studies and used to study relatedness of strains. "
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    ABSTRACT: Brucellae are tiny, aerobic, slow growing, catalase and oxidase positive Gram negative coccobacilli or small rods, which may reach man through exposure to tissues of mammalian hosts via cuts or aerosols, or as food infections mostly through dairy products. As parasites brucellae are extraordinarily successful, causing very long-lasting infections in all mammalian social animals, such as ungulates, canids, and rodents; recently they have been found to also cause disease in pinnipeds and cetaceans. Brucellae as members of the alpha Proteobacteria, have suffered major losses of genomic material as they adapted to their facultative intracellular parasite role, and are able to initiate infection with minimal disturbance of the innate immune system, thus reaching a privileged intracellular niche where they multiply. Brucellae are likely to be among the toughest organisms to control through public health and agricultural policies, even involving detection-slaughter strategies.
    Critical Reviews in Microbiology 02/2007; 33(4):325-31. DOI:10.1080/10408410701647644 · 6.02 Impact Factor
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    ABSTRACT: Objectives: Human brucellosis is a common zoonosis in Greece and an important Public Health issue. Its true incidence is likely to be higher than reported due to underreporting and misdiagnosing of chronic forms which can manifest with depression and other neuropsychiatric symptoms. The aim of this study was to investigate the possible presence of Brucella DNA in the blood of patients with emotional disorders who did not have acute brucellosis or a diagnosis of chronic brucellosis. Methods: From March 2009 to May 2009, inhabitants of a Greek region endemic for brucellosis were examined and grouped as "patients" and "controls" respectively on the basis of the presence or absence of a diagnosis of emotional disorder. A total of 78 whole blood samples were obtained and analyzed by Polymerase Chain Reaction using specific primers. Results: Brucella DNA was detected in 3 (15%) out of 20 patients with emotional disorders, whereas all controls were found to be negative. Conclusions: The appearance of psychiatric – mainly depressive – symptoms in inhabitants of brucellosis endemic areas should raise a suspicion of chronic brucellosis.
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