Kaden et al.

BMC Infectious Diseases (2017) 17:230

DOI 10.1186/s12879-017-2327-7

RESEARCH ARTICLE Open Access

A novel real-time PCR assay for specific

detection of Brucella melitensis
Rene Kaden1, Sevinc Ferrari2,3, Erik Alm4 and Tara Wahab3,4*

Abstract
Background: Brucellosis is a zoonosis that occurs worldwide. The disease has been completely eradicated in
livestock in Sweden in 1994, and all cases of confirmed human brucellosis are imported into Sweden from other
countries. However, due to an increase in the number of refugees and asylum seekers from the middle-east to
Sweden, there is a need to improve the current diagnostic methodology for Brucella melitensis. Whilst culture of
Brucella species can be used as a diagnostic tool, real-time PCR approaches provide a much faster result. The aim of
this study was to set up a species-specific real-time PCR for the detection of all biovars of Brucella melitensis, which
could be used routinely in diagnostic laboratories.
Methods: A Brucella melitensis real-time PCR assay was designed using all available genomes in the public database
of Brucella (N = 96) including all complete genomes of Brucella melitensis (N = 17). The assay was validated with a
collection of 37 Brucella species reference strains, 120 Brucella melitensis human clinical isolates, and 45 clinically
relevant non-Brucella melitensis strains.
Results: In this study we developed a single real-time PCR for the specific detection of all biovars of Brucella melitensis.
Conclusions: This new real-time PCR method shows a high specificity (100%) and a high sensitivity (1.25 GE/μl) and
has been implemented in the laboratories of four governmental authorities across Sweden.
Keywords: Brucella melitensis, Brucellosis, Real-Time PCR

Background Mediterranean basin (Portugal, Spain, Southern France,

Despite ongoing eradication programs, brucellosis is a wide- Italy, Greece, Turkey, North Africa) [3]. Brucella com-
spread zoonosis that infects mainly cattle, sheep, goats, and prises six classical species (B. abortus, B. canis, B. meliten-
pigs. It also leads to considerable financial losses in animal sis, B. neotomae, B. ovis, and B. suis) and five novel species
husbandry due to abortion and fertility problems in cattle, (B. ceti, B. microti, B. inopinata, B. papionis and B. pinni-
sheep and goats [1, 2]. Some Brucella species can also infect pedialis). B. melitensis is recognized as the main human
humans and more than 500,000 human cases are reported pathogen associated with human outbreaks worldwide [3,
annually worldwide [3]. Brucellosis is a febrile illness, some- 5, 6]. The species of the genus Brucella can be distin-
times with localized bone and tissue infection, or multi- guished on the basis of phenotype, genotype and preferred
organ disease. Transmission to humans occurs through host. Sweden was officially declared free of brucellosis in
different routes: the ingestion of unpasteurized milk and 1994 [7] and all human cases in Sweden originate from
dairy products; direct contact with infected animal tissues; countries abroad. The incidence in Sweden is between 1
or accidental ingestion, inhalation or injection of cultured and 20 reported cases per year according to the Swedish
Brucella [4]. Most human Brucellosis cases occur in Syria, National Surveillance System SmiNet-2 [8]. The gold
Turkey, Mexico and Iran, but also around the standard method for its diagnosis is the isolation of the bac-
teria from clinical samples via blood cultures and identifica-
* Correspondence: tara.wahab@folkhalsomyndigheten.se tion by classical microbiological tube testing. Brucella
3
Swedish Forum for Biopreparedness Diagnostics, Stockholm, Umeå and grows slowly and visible cultures appear after 3–4 days, but
Uppsala, Sweden it can take more than 2 weeks to obtain a definitive result
4
Department of Microbiology, The Public Health Agency of Sweden,
Stockholm, Sweden [9]. Due to its pathogenicity, a biosafety level 3 laboratory
Full list of author information is available at the end of the article

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Kaden et al. BMC Infectious Diseases (2017) 17:230 Page 2 of 6

(BSL-3) is mandatory when handling Brucella organisms. This as necessary due to the lack of necessary genome
Laboratory-acquired infections are rarely diagnosed or data in the publicly available databases at the start of this
reported, however they do occur [9, 10]. project [14]. The panel to validate the inclusivity and ex-
The application of DNA-based methods for Brucella clusivity of the real-time PCR assay contained all known
diagnosis is challenging, since all Brucella species have a biovars of Brucella, as well as 45 other non-Brucella
very high degree of genetic homology (up till 99.9%), as DNA from American Type Culture Collection (ATCC),
shown by whole genome sequencing of B. abortus, B. Culture Collection University of Gothenburg (CCUG)
melitensis, and B. suis [11–14]. However, several groups and National Collection of Type Cultures (NCTC) with
have recently developed PCR-based assays for the dis- clinical relevance. A number of environmental samples,
crimination among species and biovars of Brucella. as well as the closely related species Ochrobactrum
Three Brucella species B. abortus, B. melitensis, and B. anthropi, were also tested in this study (Tables 1 and 2).
suis have been sub-typed into biovars [15, 16]. All spe-
cies within the genus Brucella show an average similarity Bacterial strains and growth conditions
of 99% across the entire genome and a range between A collection of 31 Brucella sp. reference strains (Table 1)
93% and 99.9% based on analysis and nonparametric in- and 120 B. melitensis human clinical isolates were iso-
ference (ANI) analysis. Data from all public available lated in the BSL-3 laboratory at the FOHM, by cultiva-
complete genome sequences of all type strains and refer- tion on 5% sheep blood agar plates in a 5%–10% CO2
ence strains were included in the design of primers and atmosphere at 37 °C for 48 h. All non-Brucella strains
probes for the real-time PCR assay of this study. One were routinely cultivated on 5% sheep blood agar over
multiplex PCR (AMOS) developed by Bricker and Halling night at 37 °C.
in 1994, is applicable to differentiate between B. abortus The B. melitensis human clinical isolates were col-
biovars 1, 2 and 4, B. melitensis, and B. ovis, B. suis biovar 1 lected between 1994 and 2016 from Swedish patients,
by specific PCR products based on unique chromosomal who had returned from Brucella-endemic countries, in a
loci of the mobile genetic element IS711 in their genome biorepository of the FOHM and used as stipulated in the
[17]. This PCR was later improved by another laboratory by regulations for diagnostic development and quality as-
adding specific primers for the identification of B. abortus sessment. The FOHM performs all Brucella diagnostics
biovars 5, 6, 9 and genotype 3b of biovar 3 [18]. In 2009, on human samples in Sweden, and the B. melitensis iso-
Huber et al. developed a random amplified polymorphic lates used in this study have previously been confirmed
DNA PCR assay to differentiate all recognized Brucella by cultivation, a general real-time PCR amplifying DNA
species, including the marine mammals-infecting species B. of all Brucella strains, as well as by Matrix Assisted
ceti and B. pinnipedialis [19]. However, all published Laser Desorption/Ionization Time of Flight Mass Spec-
methods are developed to distinguish species and biovars trometry (MALDI-TOF MS) [22, 23]. Ethical review of
mainly by gel-based bar patterns and the majority of de- research involving humans is not applicable for diagnos-
scribed methods were tested with a few strains. tic development and quality assessment.
Kim et al. [20] developed a new real-time PCR for dis-
tinguishing B. abortus from other Brucella species, DNA extraction
which is based on a single nucleotide polymorphism. Bacterial DNA was extracted using the commercially
However, there was a need for a reliable real-time PCR available EZ1® DNA Tissue Kit from (Qiagen, Stockholm,
for the detection of B. melitensis at the species level [21]. Sweden) according to the protocol from the manufacturer
The method has to be validated for clinical purposes and stored at 4 °C until use. A volume of 5 μl of seal her-
with a large number of human isolates to fulfill the val- pes virus cell culture was used in each sample of the total
idation requirements of the certified laboratory of the volume of 200 μl in the extraction step as a process con-
public health institute. A Brucella genus specific real- trol. Each sample was eluted in 50 μl elution buffer.
time PCR assay is currently in use at the Public Health
Agency in Sweden (FOHM). However, B. melitensis is Bioinformatic analyses, primer & probe design and
the most prevalent species, and we need to be able to real-time PCR
differentiate to the species level, due to the epidemio- In order to identify a B. melitensis-specific target(s), all
logical significance of B. melitensis [21]. available genomes in the public database of Brucella
(N = 96) including all complete genomes of B. melitensis
Methods (N = 17) were used in the design of primers and probes.
With the aim of developing a real-time PCR assay for A 2 basepair deletion which is highly specific for B. meli-
the detection of all known species and biovars of B. tensis was found in the acetyl-CoA acetyltransferase
melitensis, the genomes of B. ceti, B. inopinata, B. neoto- gene. Primers were designed flanking this deletion and a
mae and B. suis biovar 4 were sequenced and analyzed. short 12-mer MGB probe was placed over the area with
Kaden et al. BMC Infectious Diseases (2017) 17:230 Page 3 of 6

Table 1 Reference strains tested to assess the sensitivity of The real-time PCR assays were carried out in 25 μL
Brucella melitensis specific real-time PCR assay reaction mixtures containing 5 μL template DNA, in
Species Biotype Strain PerfeCta Multiplex qPCR SuperMix (Applied Biosys-
B. melitensis 1 16 M tems®) diluted in UltraPure™ DNase/RNase-Free Distilled
1 ATCC 23456 Water (Invitrogen™), 0.9 μM of each primer, and 0.2 μM
probe. Amplification and detection were performed
1 NCTC 10094
using two different PCR machines, an ABI-7500/96-well
2 NCTC10508
plates real-time FAST PCR platform, and a StepOne
3 NCTC 10509 Plus real-time PCR system (Applied Biosystems®).
2065 Thermocycling parameters were as follows: inactivation
B. abortus 1 ATCC 23448 95 °C for 3 min, followed by 45 cycles 95 °C for 3 s, and
1 544 annealing at 60 °C for 30 s. The baseline and threshold
were set using the auto-baseline and threshold feature in
1 NCTC 00624
StepOneplus Software v2.2.2 (Applied Biosystems®). Sam-
2 NCTC10501
ples were considered positive if target amplification was
3 NCTC10502 detected within 40 cycles.
4 NCTC10503
5 NCTC10504 Determination of the real-time PCR limit of detection
6 NCTC10505 The limit of detection (LOD) was defined by using the
7 NCTC10506 Brucella ATCC 23456 strain with 106, 105, 104, 103, 102,
9 NCTC10507 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78, 0.39 genome equiva-
lents per reaction. LOD samples were analysed in six
B. suis 1 ATCC 23444
replicates for each concentration and with five runs on 5
1 NCTC 10316
days (Fig. 1).
1 NCTC 12042–01
2 NCTC 10510
Internal amplification control (IAC)
3 NCTC 10511 As internal amplification control (IAC), Phocine Herpes-
4 NC 10385–02 virus 1 (PhHV-1) aliquots with a known DNA concen-
5 NCTC 11996 tration and a target quantification cycle (Cq) of 32 was
1720 used [24].
1030
B. canis ATCC 23365 Results and discussion
Real-time PCR is a rapid and reliable method for the
SVA13
analysis of Brucella in clinical samples. However, the
NCTC 10854
high genomic similarity between different Brucella spe-
3.4.2008/122 cies makes the design of a species-specific real-time PCR
E20140122–106 assay difficult. An alternative attempt to use a probe
B. ovis ATCC 25840 (FAM-CCGCCGAGATACAAA) with Tm 68–70 °C (Pri-
NCTC 10512 mer Express v 3.0) resulted in a signal for all Brucella
species, but also a signal for non-melitensis species. To
B. ceti NCTC 12891
increase the discriminatory effect, a shorter probe
B. inopinata CAPM 6436
(FAM-CCGAGATACAAA-MGB) with Tm 57 °C was
B. microti CAPM 6434 used. By using this shorter probe we could eliminate all
B. neotomae ATCC 23459 the non-specific amplification derived from other Bru-
B. pinnipedialis NCTC 12890 cella species than B. melitensis.
The method was validated according to Broeders et
al. 2014 [25] and according to the validation standards
the deletion. The forward nucleotide sequence 5′-GC of the Swedish National Veterinary Institute (SVA), the
TCGACACAAAGGGCCA-3′ (Biomers, Germany) and Public Health Agency of Sweden (FOHM), the Swedish
the reverse nucleotide sequence 5′-CAAGCGTGGTCT National Food Agency (NFA) and the Swedish Defense
GGCGA-3′ (Biomers, Germany) were used with the FAM- Research Agency (FOI). The validation comprised ap-
labelled hydrolysis probe -CCGAGATACAAA-MGB (Ap- plicability, practicability, specificity, linearity, and
plied Biosystems®). sensitivity.
Kaden et al. BMC Infectious Diseases (2017) 17:230 Page 4 of 6

Table 2 None Brucella strains used in the study for exclusivity Table 2 None Brucella strains used in the study for exclusivity
test test (Continued)
Species Strain Taylorella equigenitalis CCUG 16464
Actinobacillus pleuropneumoniae CCUG 12837 Yersinia enterocolitica CCUG 8239
Actinomyces pyogenes CCUG 13230 Yersinia pestis 570-04
Alcaligenes denitrificans CCUG 407 Yersinia pseudotuberculosis CCUG 5855
Bacillus antracis NCTC1328
Bacillus cereus CCUG 7414 Applicability
Bacillus subtilis ATCC 6633 No false positive or false negative result is acceptable in
Bacteroides fragilis ATCC 25285 clinical BSL3 pathogen diagnostics. To guarantee a cor-
Bordetella bronchiseptica CCUG 219 rect identification of BSL3- pathogens, we strongly rec-
ommend isolation of the bacteria on selective Brucella
Burkholderia mallei NCTC120
agar plates. This enables evaluation of the phenotypic
Burkholderia pseudomallei NCTC8707
properties of the strains, such as colony morphology,
Clostridium perfringens CCUG 1795 and enriches the molecular target of the PCR, as well as
Enterococcus fecalis ATCC 29212 reduces the concentration of potential PCR inhibitors.
Erysipelotrix rhusiopatiae CCUG 221 Even after the enrichment an internal PCR process con-
Escherichia coli ATCC 35218 trol is recommended as described above. All strains in
our validation were cultured under the same conditions
Escherichia coli (EHEC) EDL333
as bacterial strains were isolated in the clinical diagnos-
Escherichia coli (VTEC) 2954-06
tics workflow. The real-time PCR method was therefore
Fusobacterium necrophorum CCUG 9994 recognized as applicable in combination with the isola-
Haemophilus influenzae ATCC 49247 tion of bacteria from clinical specimens.
Haemophilus somnus CCUG 28029
Klebsiella oxytoca CCUG 15717 Specificity
The assay was tested with 120 human clinical B. meliten-
Klebsiella pneumoniae CCUG 225
sis isolates, 31 other non B. melitensis Brucella strains,
Listeria monocytogenes CCUG 15527
45 non B. melitensis strains, and 6 B. melitensis refer-
Nocardia asteroides CCUG 10073 ence strains. There was no amplification from any other
Ochrabactrum anthropi ATCC 49188 Brucella species other than B. melitensis. The specificity
Pasteurella multocida CCUG 229 of this method was 100% because all 126 B. melitensis
Pasteurella pneumotropica CCUG 12398 samples were tested positive while all 76 non-B. meliten-
sis samples gave no amplification in the real-time PCR.
Proteus mirabilis CCUG 26767
Pseudomonas aeruginosa CCUG 17619
Practicability
Rhodococcus equi CCUG 892 The assay was used for the identification of Brucella
Salmonella Dublin CCUG 35631 samples from the first External Quality Assurance Exer-
Salmonella Thyphimurium CCUG 31969 cises (EQAEs) on highly infectious agents (BSL-3) from
Salmonella Zanzibar CCUG 41921 the EU financed project “Efficient response to highly
dangerous and emerging pathogens” (EMERGE). The re-
Staphylococcus aureus CCUG 4151
sults obtained with this assay conformed to the results
Staphylococcus intermedius CCUG 49053
of other laboratories.
Streptobacillus moniliformis CCUG 33440
Streptococcus agalactiae CCUG 39325 Sensitivity
Streptococcus dysgalactiae CCUG 27436 The limit of detection was 6.25 genome equivalents of B.
Streptococcus equi CCUG 27367 melitensis (ATCC 23456) per reaction of template DNA
(1.25 GE/μl). At a DNA concentration of 3.125 genome
Streptococcus pyogenes CCUG 12701
equivalents per reaction, 80% of the reactions were still
Streptococcus uberis CCUG 27444
positive (Fig. 1).
Streptococcus zooepidemicus CCUG 23256
Linearity
The linear dynamic range of this PCR assay was deter-
mined by testing serially diluted DNA of B. melitensis
Kaden et al. BMC Infectious Diseases (2017) 17:230 Page 5 of 6

Fig. 1 Limit of detection was determined by assaying six replicates of ten and two fold serially diluted DNA of strain Brucella melitensis ATCC
23456 in five separate experiments. The number of positives per total number of replicates tested is shown in the figure

strain ATCC 23456. The regression coefficient calculated We thank Finnish Food Safety Authority Evira for sending us their two
from the regression line in the standard curve was Brucella canis isolates 3.4.2008/122 and E2014122-106.

R2 = 0.993, y-intercept = 37.766.

Funding
In conclusion, the novel real-time PCR assay is highly This project is part of ongoing work to develop and harmonise methods to
sensitive and reliable for the rapid detection of all bio- increase the level of biopreparedness in Sweden and is performed within
vars of B. melitensis. It is an important tool for diagnos- the Forum for Biopreparedness Diagnostics (FBD), involving four governmental
institutes: the National Veterinary Institute (SVA), the Public Health Agency of
tic laboratories for clinical samples from both humans Sweden (FOHM), the National Food Agency (NFA), and the Swedish Defence
and animals. Research Agency (FOI) financed by by the Swedish Civil Contingencies
Agency. The new real-time PCR method was also tested at the Swedish La-
boratory for Food Safety and Biopreparedness (RUB), as a collaborative effort of
Conclusions the SVA and the NFA.
This new real-time PCR method for the detection of all
Availability of data and materials
biovars of B. melitensis shows a high specificity (100%) All available genomes in the public database of Brucella (N = 96), including
and a high sensitivity (1.25 GE/μl) and has been success- all complete genomes of B. melitensis (N = 17), were used in the design of
fully implemented in laboratories of four governmental au- primers and probes in August 2016.
thorities across Sweden (National Food Agency, National
Authors’ contributions
Veterinary Institute, Swedish Defence Research Agency EA carried out the sequence analysis, RK participated in the design of the
and The Public Health Agency of Sweden). study and drafted the manuscript. SF carried out the exclusivity test. TW
conceived the study, participated in its design, drafted the manuscript, and
Abbreviations performed most of the laboratory work. All authors read and approved the
ANI: Analysis and nonparametric inference; ATCC: American type culture final manuscript.
collection; B: Brucella; BLAST: Basic local alignment search tool; BSL: Biosafety
level; CCUG: Culture Collection University of Göteborg; Cq: Quantification Competing interests
cycle; DNA: Deoxyribonucleic acid; EMERGE: Efficient response to highly The authors declare that they have no competing interests.
dangerous and emerging pathogens; EQAE: External quality assurance exercises;
FAM: 6-carboxyfluorescein; FOHM: Public Health Agency of Sweden; IAC: Internal Consent for publication
amplification control; LOD: Limit of detection; MALDI-TOF MS: Matrix assisted Not applicable.
laser dessorption/Ionization time of flight mass spectrometry; MGB: Minor
groove binder; NCBI: National Center for Biotechnology Information;
Ethics approval and consent to participate
NCTC: National collection of type cultures; NFA: National food agency;
Ethical Review of Research Involving Humans is not applicable for diagnostic
PCR: Polymerase chain reaction; PhHV-1: Phocine herpesvirus 1; SVA: National
development and quality assessment. In this study we used an historical
Veterinary Institute; Tm: Melting temperature
collection of Brucella from the Public Health Agency of Sweden. No data from
human patients were used, therefore informed consent was not required.
Acknowledgements
The authors gratefully thank Martina Lindberg, Stina Bäckman and Talar
Boskani for technical assistance, Nina Lagerqvist, Annelie Lundin Zumpe, Publisher’s Note
Anna-Lena Johansson and Moa Lavander for fruitful discussion, Öjar Melfors, Springer Nature remains neutral with regard to jurisdictional claims in published
Romanico Arrighi and Leigh Davidsson for critical reading of the manuscript. maps and institutional affiliations.
Kaden et al. BMC Infectious Diseases (2017) 17:230 Page 6 of 6

Author details 20. Kim J-Y, Kang S-I, Lee JJ, Lee K, Sung S-R, Erdenebaataar J, Vanaabaatar B,
1
Department of Medical Sciences, Clinical Microbiology, Uppsala University, Jung SC, Park YH, Yoo H-S, et al. Differential diagnosis of Brucella abortus by
Uppsala, Sweden. 2National Veterinary Institute, Uppsala, Sweden. 3Swedish real-time PCR based on a single-nucleotide polymorphisms. J Vet Med Sci.
Forum for Biopreparedness Diagnostics, Stockholm, Umeå and Uppsala, 2016;78(4):557–62.
Sweden. 4Department of Microbiology, The Public Health Agency of Sweden, 21. Andriopoulos P, Tsironi M. Molecular Diagnosis of Brucellosis: A Brief Report.
Stockholm, Sweden. Adv Mol Diagn. 2016;1(2):108-10.
22. Drevinek M, Dresler J, Klimentova J, Pisa L, Hubalek M. Evaluation of sample
Received: 16 November 2016 Accepted: 16 March 2017 preparation methods for MALDI-TOF MS identification of highly dangerous
bacteria. Lett Appl Microbiol. 2012;55(1):40–6.
23. Lista F, Reubsaet FA, De Santis R, Parchen RR, de Jong AL, Kieboom J, van der
Laaken AL, Voskamp-Visser IA, Fillo S, Jansen HJ, et al. Reliable identification at
References the species level of Brucella isolates with MALDI-TOF-MS. BMC Microbiol.
1. Pappas G, Akritidis N, Tsianos EV. Brucellosis - Reply. New Engl J Med. 2005; 2011;11:267.
353(10):1072. 24. van Doornum GJJ, Guldemeester J, Osterhaus ADME, Niesters HGM.
2. Seleem MN, Boyle SM, Sriranganathan N. Brucellosis: a re-emerging Diagnosing Herpesvirus Infections by Real-Time Amplification and Rapid
zoonosis. Vet Microbiol. 2010;140(3–4):392–8. Culture. J Clin Microbiol. 2003;41(2):576–80.
3. Pappas G, Akritidis N, Bosilkovski M, Tsianos E. Brucellosis. N Engl J Med. 25. Broeders S, Huber I, Grohmann L, Berben G, Taverniers I, Mazzara M, Roosens
2005;352(22):2325–36. N, Morisset D. Guidelines for validation of qualitative real-time PCR methods.
4. Young EJ. Human brucellosis. Rev Infect Dis. 1983;5(5):821–42. Trends Food Sci Technol. 2014;37(2):115–26.
5. Whatmore AM. Current understanding of the genetic diversity of Brucella,
an expanding genus of zoonotic pathogens. Infect Genet Evol. 2009;9(6):
1168–84.
6. Whatmore AM, Davison N, Cloeckaert A, Al Dahouk S, Zygmunt MS, Brew
SD, Perrett LL, Koylass MS, Vergnaud G, Quance C, et al. Brucella papionis sp.
nov., isolated from baboons (Papio spp.). Int J Syst Evol Microbiol. 2014;
64(Pt 12):4120–8.
7. European Commission. Commission Decision of 23 June 2003 establishing
the official tuberculosis, brucellosis, and enzootic-bovine-leukosis-free status
of certain Member States and regions of Member States as regards bovine
herds (Text with EEA relevance) (notified under document number C(2003).
EC Official J. 2003;(L156):0074-0078.
8. Rolfhamre P, Jansson A, Arneborn M, Ekdahl K. SmiNet-2: Description of an
internet-based surveillance system for communicable diseases in Sweden.
Euro Surveill. 2006;11(5):103–7.
9. Al Dahouk S, Tomaso H, Nockler K, Neubauer H, Frangoulidis D. Laboratory-
based diagnosis of brucellosis–a review of the literature. Part II: serological
tests for brucellosis. Clin Lab. 2003;49(11–12):577–89.
10. Garofolo G, Fasanella A, Di Giannatale E, Platone I, Sacchini L, Persiani T,
Boskani T, Rizzardi K, Wahab T. Cases of human brucellosis in Sweden linked
to Middle East and Africa. BMC Res Notes. 2016;9(1):277.
11. DelVecchio VG, Kapatral V, Redkar RJ, Patra G, Mujer C, Los T, Ivanova N,
Anderson I, Bhattacharyya A, Lykidis A, et al. The genome sequence of the
facultative intracellular pathogen Brucella melitensis. Proc Natl Acad Sci U S
A. 2002;99(1):443–8.
12. Halling SM, Peterson-Burch BD, Bricker BJ, Zuerner RL, Qing Z, Li LL, Kapur V,
Alt DP, Olsen SC. Completion of the genome sequence of Brucella abortus
and comparison to the highly similar genomes of Brucella melitensis and
Brucella suis. J Bacteriol. 2005;187(8):2715–26.
13. Paulsen IT, Seshadri R, Nelson KE, Eisen JA, Heidelberg JF, Read TD, Dodson
RJ, Umayam L, Brinkac LM, Beanan MJ, et al. The Brucella suis genome
reveals fundamental similarities between animal and plant pathogens and
symbionts. Proc Natl Acad Sci U S A. 2002;99(20):13148–53.
14. Wahab T, Ferrari S, Lindberg M, Backman S, Kaden R. Draft Genome Sequences
of Brucella suis Biovar 4 Strain NCTC 10385, Brucella ceti Strain NCTC 12891T,
Brucella inopinata Strain CAMP 6436T, and Brucella neotomae Strain ATCC
23459T. Genome Announc. 2014;2(5).
15. Garcia-Yoldi D, Marin CM, de Miguel MJ, Munoz PM, Vizmanos JL, Lopez-
Goni I. Multiplex PCR assay for the identification and differentiation of all Submit your next manuscript to BioMed Central
Brucella species and the vaccine strains Brucella abortus S19 and RB51 and and we will help you at every step:
Brucella melitensis Rev1. Clin Chem. 2006;52(4):779–81.
16. Yu WL, Nielsen K. Review of detection of Brucella spp. by polymerase chain • We accept pre-submission inquiries
reaction. Croat Med J. 2010;51(4):306–13. • Our selector tool helps you to find the most relevant journal
17. Bricker BJ, Halling SM. Differentiation of Brucella abortus bv. 1, 2, and 4,
• We provide round the clock customer support
Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. J Clin
Microbiol. 1994;32(11):2660–6. • Convenient online submission
18. Ocampo-Sosa AA, Aguero-Balbin J, Garcia-Lobo JM. Development of a new • Thorough peer review
PCR assay to identify Brucella abortus biovars 5, 6 and 9 and the new
• Inclusion in PubMed and all major indexing services
subgroup 3b of biovar 3. Vet Microbiol. 2005;110(1–2):41–51.
19. Huber B, Scholz HC, Lucero N, Busse HJ. Development of a PCR assay • Maximum visibility for your research
for typing and subtyping of Brucella species. Int J Med Microbiol. 2009;
299(8):563–73. Submit your manuscript at
www.biomedcentral.com/submit