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Advancement of a multiplex PCR for the differentiation of all currently described Brucella species
Journal of Microbiological Methods 80 (2010) 112–114
Contents lists available at ScienceDirect
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 b
Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1, 12277 Berlin, Germany Bundeswehr Institute of Microbiology, Munich, Germany
a r t i c l e
i n f o
Article history: Received 16 July 2009 Received in revised form 26 October 2009 Accepted 27 October 2009 Available online 1 November 2009
a b s t r a c t To facilitate routine laboratories in the effective diagnosis of brucellosis, we report a robust and rapid multiplex PCR assay, which allows for the differentiation of all nine currently recognised Brucella species. This includes the recently described species B. microti, B. inopinata, B. ceti and B. pinnipedialis. © 2009 Elsevier B.V. All rights reserved.
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 Brucella 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: [email protected] (A. Mayer-Scholl). 0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.10.015
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
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, Brucellaceae (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.
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A. Mayer-Scholl et al. / Journal of Microbiological Methods 80 (2010) 112–114
References Bricker, B.J., 2002. PCR as a diagnostic tool for brucellosis. Vet. Microbiol. 90, 435–446. Bricker, B.J., Halling, S.M., 1994. Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. J. Clin. Microbiol. 32, 2660–2666. De, B.K., Stauffer, L., Koylass, M.S., Sharp, S.E., Gee, J.E., Helsel, L.O., Steigerwalt, A.G., Vega, R., Clark, T.A., Daneshvar, M.I., Wilkins, P.P., Whatmore, A.M., 2008. Novel Brucella strain (BO1) associated with a prosthetic breast implant infection. J. Clin. Microbiol. 46, 43–49. Foster, G., Jahans, K.L., Reid, R.J., Ross, H.M., 1996. Isolation of Brucella species from cetaceans, seals and an otter. Vet. Rec. 138, 583–586. Garcia-Yoldi, D., Marin, C.M., de Miguel, M.J., Munoz, P.M., Vizmanos, J.L., Lopez-Goni, I., 2006. Multiplex PCR assay for the identification and differentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1. Clin. Chem. 52, 779–781. Hinic, V., Brodard, I., Thomann, A., Cvetnic, Z., Makaya, P.V., Frey, J., Abril, C., 2008. 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. J. Microbiol. Methods 75, 375–378.
Jahans, K.L., Foster, G., Broughton, E.S., 1997. The characterisation of Brucella strains isolated from marine mammals. Vet. Microbiol. 57, 373–382. Scholz, H.C., Hubalek, Z., Sedlacek, I., Vergnaud, G., Tomaso, H., Al Dahouk, S., Melzer, F., Kampfer, P., Neubauer, H., Cloeckaert, A., Maquart, M., Zygmunt, M.S., Whatmore, A.M., Falsen, E., Bahn, P., Gollner, C., Pfeffer, M., Huber, B., Busse, H.J., Nockler, K., 2008. Brucella microti sp. nov., isolated from the common vole Microtus arvalis. Int. J. Syst. Evol. Microbiol. 58, 375–382. Scholz, H.C., Nöckler, K., Göllner, C., Bahn, P., Vergnaud, G., Tomaso, H., Al Dahouk, S., Kämpfer, P., Cloeckaert, A., Maquart, M., Zygmunt, M.S., Whatmore, A., Pfeffer, M., Huber, B., Busse, B.-J., De, B.K., 2009. Brucella inopinata sp. nov., isolated from a breast implant infection. Int. J. Syst. Evol. Microbiol. Sohn, A.H., Probert, W.S., Glaser, C.A., Gupta, N., Bollen, A.W., Wong, J.D., Grace, E.M., McDonald, W.C., 2003. Human neurobrucellosis with intracerebral granuloma caused by a marine mammal Brucella spp. Emerg. Infect. Dis. 9, 485–488. Velasco, J., Romero, C., Lopez-Goni, I., Leiva, J., Diaz, R., Moriyon, I., 1998. Evaluation of the relatedness of Brucella spp. and Ochrobactrum anthropi and description of Ochrobactrum intermedium sp. nov., a new species with a closer relationship to Brucella spp. Int. J. Syst. Bacteriol. 48 (Pt 3), 759–768. Verger, J.M., Grimont, F., Grimont, P.A.D., Grayon, M., 1985. Brucella, a monospecific genus as shown by deoxyribonucleic-acid hybridization. Int. J. Syst. Bacteriol. 35, 292–295.
Contents lists available at ScienceDirect
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 b
Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1, 12277 Berlin, Germany Bundeswehr Institute of Microbiology, Munich, Germany
a r t i c l e
i n f o
Article history: Received 16 July 2009 Received in revised form 26 October 2009 Accepted 27 October 2009 Available online 1 November 2009
a b s t r a c t To facilitate routine laboratories in the effective diagnosis of brucellosis, we report a robust and rapid multiplex PCR assay, which allows for the differentiation of all nine currently recognised Brucella species. This includes the recently described species B. microti, B. inopinata, B. ceti and B. pinnipedialis. © 2009 Elsevier B.V. All rights reserved.
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 Brucella 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: [email protected] (A. Mayer-Scholl). 0167-7012/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2009.10.015
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
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, Brucellaceae (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.
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References Bricker, B.J., 2002. PCR as a diagnostic tool for brucellosis. Vet. Microbiol. 90, 435–446. Bricker, B.J., Halling, S.M., 1994. Differentiation of Brucella abortus bv. 1, 2, and 4, Brucella melitensis, Brucella ovis, and Brucella suis bv. 1 by PCR. J. Clin. Microbiol. 32, 2660–2666. De, B.K., Stauffer, L., Koylass, M.S., Sharp, S.E., Gee, J.E., Helsel, L.O., Steigerwalt, A.G., Vega, R., Clark, T.A., Daneshvar, M.I., Wilkins, P.P., Whatmore, A.M., 2008. Novel Brucella strain (BO1) associated with a prosthetic breast implant infection. J. Clin. Microbiol. 46, 43–49. Foster, G., Jahans, K.L., Reid, R.J., Ross, H.M., 1996. Isolation of Brucella species from cetaceans, seals and an otter. Vet. Rec. 138, 583–586. Garcia-Yoldi, D., Marin, C.M., de Miguel, M.J., Munoz, P.M., Vizmanos, J.L., Lopez-Goni, I., 2006. Multiplex PCR assay for the identification and differentiation of all Brucella species and the vaccine strains Brucella abortus S19 and RB51 and Brucella melitensis Rev1. Clin. Chem. 52, 779–781. Hinic, V., Brodard, I., Thomann, A., Cvetnic, Z., Makaya, P.V., Frey, J., Abril, C., 2008. 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. J. Microbiol. Methods 75, 375–378.
Jahans, K.L., Foster, G., Broughton, E.S., 1997. The characterisation of Brucella strains isolated from marine mammals. Vet. Microbiol. 57, 373–382. Scholz, H.C., Hubalek, Z., Sedlacek, I., Vergnaud, G., Tomaso, H., Al Dahouk, S., Melzer, F., Kampfer, P., Neubauer, H., Cloeckaert, A., Maquart, M., Zygmunt, M.S., Whatmore, A.M., Falsen, E., Bahn, P., Gollner, C., Pfeffer, M., Huber, B., Busse, H.J., Nockler, K., 2008. Brucella microti sp. nov., isolated from the common vole Microtus arvalis. Int. J. Syst. Evol. Microbiol. 58, 375–382. Scholz, H.C., Nöckler, K., Göllner, C., Bahn, P., Vergnaud, G., Tomaso, H., Al Dahouk, S., Kämpfer, P., Cloeckaert, A., Maquart, M., Zygmunt, M.S., Whatmore, A., Pfeffer, M., Huber, B., Busse, B.-J., De, B.K., 2009. Brucella inopinata sp. nov., isolated from a breast implant infection. Int. J. Syst. Evol. Microbiol. Sohn, A.H., Probert, W.S., Glaser, C.A., Gupta, N., Bollen, A.W., Wong, J.D., Grace, E.M., McDonald, W.C., 2003. Human neurobrucellosis with intracerebral granuloma caused by a marine mammal Brucella spp. Emerg. Infect. Dis. 9, 485–488. Velasco, J., Romero, C., Lopez-Goni, I., Leiva, J., Diaz, R., Moriyon, I., 1998. Evaluation of the relatedness of Brucella spp. and Ochrobactrum anthropi and description of Ochrobactrum intermedium sp. nov., a new species with a closer relationship to Brucella spp. Int. J. Syst. Bacteriol. 48 (Pt 3), 759–768. Verger, J.M., Grimont, F., Grimont, P.A.D., Grayon, M., 1985. Brucella, a monospecific genus as shown by deoxyribonucleic-acid hybridization. Int. J. Syst. Bacteriol. 35, 292–295.