Journal of Microbiological Methods 80 (2010) 112–114

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Journal of Microbiological Methods

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h

Note

Advancement of a multiplex PCR for the differentiation of all currently described

Brucella species
Anne Mayer-Scholl a,⁎, Angelika Draeger a, Cornelia Göllner a, Holger C. Scholz b, Karsten Nöckler a
a
Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1, 12277 Berlin, Germany
b
Bundeswehr Institute of Microbiology, Munich, Germany

a r t i c l e i n f o a b s t r a c t

Article history: To facilitate routine laboratories in the effective diagnosis of brucellosis, we report a robust and rapid
Received 16 July 2009 multiplex PCR assay, which allows for the differentiation of all nine currently recognised Brucella species.
Received in revised form 26 October 2009 This includes the recently described species B. microti, B. inopinata, B. ceti and B. pinnipedialis.
Accepted 27 October 2009
© 2009 Elsevier B.V. All rights reserved.
Available online 1 November 2009

Keywords:
Brucella
Multiplex PCR
Diagnostics
Brucellosis

Brucellosis is a re-emerging zoonosis caused by the closely related
species of the genus Brucella. The genus consists of 6 classic species, of
which B. melitensis, B. abortus, B. suis and B. canis are pathogenic for
humans. B. ceti and B. pinnipedialis are species isolated from sea
mammals (Foster et al., 1996; Jahans et al., 1997) and can occasionally
cause disease in man (Sohn et al., 2003). More recently a novel species
(B. microti) isolated from the common vole (Microtus arvalis) has been
approved (Scholz et al., 2008) and another new species has been
described from a human breast implant (B. inopinata) (De et al., 2008;
Scholz et al., 2009).
Although sensitive and very rapid, DNA-based techniques for Bru-
cella species identification are challenging due to the high genetic
interspecies homology, which exceeds 90% (Verger et al., 1985).
Published protocols include the AMOS PCR (Bricker, 2002; Bricker and
Halling, 1994), which differentiates four Brucella species, i.e. B. abortus
(biovar 1, 2, and 4), B. melitensis (biovar 1, 2, and 3), B. ovis and B. suis
(biovar 1) and a PCR assay suitable for conventional and real time
applications, which discriminates B. abortus, B. suis, B. ovis, B. canis, and
B. neotomae (Hinic et al., 2008).
Garcia-Yoldi et al. (2006)) developed a multiplex PCR for the
identification of all B. abortus biotypes, for B. melitensis, B. ovis, B. canis,
B. neotomae, B. suis, for Brucella spp. isolated from marine mammals
and for some vaccine strains. However, this multiplex PCR cannot
differentiate B. microti from B. suis and was neither tested for the
identification of B. inopinata nor for the differentiation between the two
marine mammal Brucella species, B. ceti and B. pinnipedialis. Here we
⁎ Corresponding author.
E-mail address: anne.mayer-scholl@bfr.bund.de (A. Mayer-Scholl).
report the advancement of the Garcia-Yoldi protocol, which allows the
accurate differentiation of all currently known Brucella species.
The DNA of 22 Brucella reference strains and 59 field isolates
including all currently known species and biotypes were used for the
evaluation of this multiplex PCR. The strains were provided by the
Federal Institute for Risk Assessment (Germany), the Institut National
de la Recherche Agronomique (France) and the Bundeswehr Institute
of Microbiology (Germany). B. ceti DNA was kindly provided by A.
Cloeckaert from INRA Infectiologie Animale et Santé Publique, France.
The PCR was further tested with 33 potentially serologically cross
reactive and phylogenetically related bacteria.
DNA was extracted with the QIAamp DNA Mini Kit after
inactivation for 2 h at 80 °C. The 8 PCR primer pairs described by
Garcia-Yoldi et al. were used. Further, the primer pair identifying B.
microti (Scholz et al., 2008) was included and the PCR assay adjusted
to the following parameters: a 25 µl reaction mixture containing
12.5 µl 2× Qiagen Multiplex PCR Mastermix, 2.5 µl primer mix (each
primer 2 pmol/µl) and 1 µl template DNA was utilised. Thermal
cycling was carried out with the 2720 Thermal Cycler (Applied
Biosystems). An initial denaturation step at 95 °C for 15 min was
followed by template denaturation at 94 °C for 30 s, primer annealing
at 58 °C for 90 s, a 3 min primer extension at 72 °C for a total of
25 cycles, with a subsequent final extension phase of 10 min at 72 °C.
The PCR products were analysed with a 1.5% agarose gel.
An example of the modified multiplex PCR is presented in Fig. 1. All
Brucella species including their biotypes show the band profile as
formerly described (Garcia-Yoldi et al., 2006). The additional B. microti
primer pair amplifies a distinguishing 510 bp fragment in addition to
the seven fragments (1682 bp, 1071 bp, 794 bp, 587 bp, 450 bp, 272 bp,
and 152 bp) found in B. suis in the original multiplex PCR, enabling clear

0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2009.10.015
 A. Mayer-Scholl et al. / Journal of Microbiological Methods 80 (2010) 112–114 113

Fig. 1. Identification and differentiation of all known Brucella species and biotypes by modified multiplex PCR assay. Lanes 1–8: B. abortus biotypes 1–7 and biotype 9; lanes 9–13:
B. suis biotypes 1–5; lanes 14–16: B. melitensis biotypes 1–3; lane 17: B. ovis; lane 18: B. canis; lane 19: B. neotomae; lane 20: B. pinnipedialis; lane 21: B. ceti; lane 22: B. microti;
lane 23: B. inopinata.

differentiation of B. suis and B. microti. Both the B. microti reference
strain and the five B. microti field isolates give a consistent profile.
The newly described B. inopinata strain band profile was most
similar to the B. abortus profile (152 bp, 450 bp, 587 bp, 794 bp, and
1682 bp) but can be distinguished by the presence of an additional
272 bp fragment.
In the original work by Garcia-Yoldi et al. the two marine mammal
Brucella species, B. ceti and B. pinnipedialis could not be differentiated.
In the modified protocol a slight size difference of the 794 bp fragment
was noted in the agarose gel between the two species (Fig. 2). The PCR
products were repeatedly sequenced and sequence analyses performed
(Laser Gene). A partial duplication of a 19 bp segment was found in the
B. ceti strain, of which a 15 bp large region was 100% homologous to the
neighbouring repetitive sequence (data not shown). The size difference
of the 794 bp product was demonstrated for all tested B. ceti and B.
pinnipedialis field isolates. With the exception of B. canis, the 794 bp
fragment is amplified in all Brucella strains. B. suis, B. neotomae, B.
microti, B. pinnipedialis and B. inopinata exhibit the 19 bp additional
sequence which does not appear for B. abortus, B. melitensis and B. ovis
and B. ceti (Fig. 2).
Of the 81 Brucella samples tested, all reference strains and field
isolates displayed the correct band profile. All 33 non-Brucella strains
tested negative with the described multiplex PCR. This included 6
different species of the genus Ochrobactrum, which is genetically closely
related to the genus Brucella and belongs to the same family, Brucella-
ceae (Velasco et al., 1998).
Here we introduce important supplements to a previously
described Brucella multiplex PCR. This assay can now be applied for
the identification of all currently known Brucella strains and their
biotypes in the same test. This includes distinguishing between the
marine species B. ceti and B. pinnipedialis and correctly identifying the
recently described species B. microti and B. inopinata. This rapid and
robust multiplex PCR system is an important tool for routine
laboratories for the effective diagnosis of brucellosis.
Acknowledgment
This work was supported by the project ‘Brucellenkontam’ (No.
07HS022) of the German Federal Ministry of Food, Agriculture and
Consumer Protection.

Fig. 2. Presentation of the size difference of the 794 bp PCR product. B. abortus, B. melitensis, B. ovis and B. ceti are characterised by a slightly smaller PCR fragment than B. suis,
B. neotomae, B. pinnipedialis, B. microti and B. inopinata. The 794 bp band is not amplified in the B. canis species.
114 A. Mayer-Scholl et al. / Journal of Microbiological Methods 80 (2010) 112–114
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