Skip to main content

Advertisement

SpringerLink
Log in

Evaluation of eryC as a Molecular Marker for the Quantitative Detection of Brucella Spp. by Real-Time PCR in Food Samples

  • Published:
Food Analytical Methods Aims and scope Submit manuscript

Abstract

We evaluated the capacity of the Brucella sp. eryC gene as a diagnostic marker for brucellosis by quantitative real-time PCR. eryC gene encodes the enzyme d-erythrulose-1-phosphate dehydrogenase that plays an important role in the erythritol metabolism and is related with the Brucella survival in the intracellular environment of the macrophage. The assay includes an internal amplification control (IAC) in order to avoid false negative results. It was 100% specific, with an analytical sensitivity of 1 genome equivalent (GE) in 43% of the reactions, being the quantification highly linear (R 2 > 0.9953) and efficient (PCR efficiency >0.8820) over a 6-log dynamic range, down to 10 GE. Finally, the applicability of this assay was evaluated with artificially contaminated biological matrices implicated in the transmission of this bacterium such as sheep raw milk and pig blood. The eryC-IAC real-time PCR assay allowed detection of as few as ten Brucella cells per 25 ml of sheep raw milk or per 1 ml of pig blood. In conclusion, we present an alternative for the detection of Brucella genus and therefore facilitate the establishment of preventive and prophylactic measures in food and farm environments.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Al Dahouk S, Nöckler K, Scholz HC, Pfeffer M, Neubauer H, Tomaso H (2007) Evaluation of genus-specific and species-specific real-time PCR assays for the identification of Brucella spp. Clin Chem Lab Med 45:1464–1470

    Article  CAS  Google Scholar 

  • Anderson JD, Smith H (1965) The metabolism of erythritol by Brucella abortus. J Gen Microbiol 38:109–124

    Article  CAS  Google Scholar 

  • Anonymous (1999) Sequence detection systems quantitative assay design and optimization. PE Biosystems, Foster City, CA, USA

    Google Scholar 

  • Anonymous (2016) Terrestrial Code, 25th Edition. World Organisaiton for Animal Health. ISBN: 978-92-95108-01-1

  • Baily GG, Krahn JB, Drasar BS, Stoker NG (1992) Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg 95:271–275

    CAS  Google Scholar 

  • Blasco JM, Marín C, Jiménez de Bagués M, Barberán M, Hernández A, Molina L, Velasco J, Díaz R, Morrión I (1994) Evaluation of allergic and serological tests for diagnosing Brucella melitensis infection in sheep. J Clin Microbiol 32:1835–1840

    CAS  Google Scholar 

  • Bogdanovich T, Skurnik M, Lübeck PS, Ahrens P, Hoorfar J (2004) Validated 5 nuclease PCR assay for rapid identification of the genus Brucella. J Clin Microbiol 42:2261–2263

    Article  CAS  Google Scholar 

  • Bounaadja L, Albert D, Chénais B, Hénault S, Zygmunt MS, Poliak S, Garin-Bastuji B (2009) Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes. Vet Microbiol 137:156–164

    Article  CAS  Google Scholar 

  • Burkhardt S, De Bagües MPJ, Liautard JP, Köhler S (2009) Analysis of the behavior of eryC mutants of Brucella suis attenuated in macrophages. Infect Immun 73:6782–6790

    Article  Google Scholar 

  • Da Costa M, Guillou JP, Garin-Bastuji B, Thiébaud M, Dubray G (1996) Specificity of six gene sequences for the detection of the genus Brucella by DNA amplification. J Appl Bacteriol 81:267–275

    Article  CAS  Google Scholar 

  • Debeaumont C, Falconnet PA, Maurin M (2005) Real-time PCR for detection of Brucella spp. DNA in human serum samples. Eur J Microbiol Infect Dis 24:842–845

    Article  CAS  Google Scholar 

  • Fekete A, Bantle JA, Halling SM, Sanborn MR (1990) Preliminary development of a diagnostic test for Brucella using polymerase chain reaction. J Appl Bacteriol 69:216–227

    Article  CAS  Google Scholar 

  • Herman L, De Ridder H (1992) Identification of Brucella spp. by using the polymerase chain reaction. Appl Environ Microbiol 58:2099–2101

    CAS  Google Scholar 

  • Hinic V, Brodard I, Thomann A, Cvetnic Z, Makaya PV, Frey J, Abril C (2000) 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

    Article  Google Scholar 

  • Hinic V, Brodard I, Thomann A, Holub M, Miserez R, Abril C (2009) IS711-based real-time PCR assay as a tool for detection of Brucella spp. in wild boars and comparison with bacterial isolation and serology. BMC Vet Res 5:2

  • Leal-Klevezas DS, Martínez-Vázquez IO, López-Merino A, Martínez-Soriano JP (1995) Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. J Clin Microbiol 33:3087–3090

    CAS  Google Scholar 

  • Leal-Klevezas DS, Martínez-Vázquez IO, García-Cantú J, López-Merino A, Martínez-Soriano JP (2006) Use of polymerase chain reaction to detect Brucella abortus biovar 1 in infected goats. Vet Microbiol 75:91–97

    Article  Google Scholar 

  • López-Enríquez L, Rodríguez-Lázaro D, Hernández M (2007) Quantitative detection of Clostridium tyrobutyricum in milk by real-time PCR. Appl Environ Microbiol 73:3747–3751

    Article  Google Scholar 

  • Marianelli C, Martucciello A, Tarantino M, Vecchio R, Iovane G, Galiero G (2008) Evaluation of molecular methods for the detection of Brucella species in water buffalo milk. J Dairy Sci 91:3779–3786

    Article  CAS  Google Scholar 

  • Morata P, Queipo-Ortuño MI, Colmenero JD (1998) Strategy for optimizing DNA amplification in a peripheral blood PCR assay used for diagnosis of human brucellosis. J Clin Microbiol 36:2443–2446

    CAS  Google Scholar 

  • Navarro E, Escribano J, Fernández JA, Solera J (1998) Comparison of three different PCR methods for detection of Brucella spp. in human blood samples. FEMS Immunol Med Microbiol 34:147–151

    Article  Google Scholar 

  • Navarro E, Casao MA, Solera J (2004) Diagnosis of human brucellosis. Expert Rev Mol Diagn 4:115–123

    Article  CAS  Google Scholar 

  • Navarro E, Segura JC, Castaño MJ, Solera J (2006) Use of real-time quantitative polymerase chain reaction to monitor the evaluation of Brucella melitensis DNA load during therapy and post-therapy follow-up in patients with brucellosis. Clin Infect Dis 42:1266–1273

    Article  CAS  Google Scholar 

  • O’Leary S, Sheahan M, Sweeney T (2006) Brucella abortus detection by PCR assay in blood, milk and lymph tissue of serologically positive cows. Res Vet Sci 81:170–176

    Article  Google Scholar 

  • Ocampo-Sosa AA, Agüero Balbín J, García Lobo JM (2005) Development of a new PCR assay to identify Brucella abortus biovars 5, 6 and 9 and the new subgroup 3b of biovar 3. Vet Microbiol 110:41–51

    Article  CAS  Google Scholar 

  • Oravcová K, López-Enríquez L, Rodríguez-Lázaro D, Hernández M (2009) Mycoplasma agalactiae p40 Gene, a novel marker for diagnosis of contagious agalactia in sheep by real-time PCR: assessment of analytical performance and in-house validation using naturally contaminated milk samples. J Clin Microbiol 47:445–450

    Article  Google Scholar 

  • Probert WS, Scharader KN, Khuong NY, Bystrom SL, Graves MH (2004) Real-time multiplex PCR assay for detection of Brucella spp., B. abortus, and B. melitensis. J Clin Microbiol 42:1290–1293

    Article  CAS  Google Scholar 

  • Queipo-Ortuño MI, Colmenero JD, Reguera JM, García-Ordoñez MA, Pachón ME, Gonzalez M, Morata P (2005) Rapid diagnosis of human brucellosis by SYBR Green I-based real-time PCR assay and melting curve analysis in serum samples. Clin Microbiol Infect 11:713–718

    Article  Google Scholar 

  • Queipo-Ortuño MI, Colmenero JD, Bravo MJ, García-Ordoñez MA, Morata P (2008) Usefulness of a quantitative real-time PCR assay using serum samples to discriminate between inactive, serologically positive and active human brucellosis. Clin Microbiol Infect 14:1128–1134

    Article  Google Scholar 

  • Redkar R, Rose S, Bricker B, DelVecchio V (2001) Real-time detection of Brucella abortus, Brucella melitensis and Brucella suis. Mol Cell Probes 15:43–52

    Article  CAS  Google Scholar 

  • Rodríguez-Lázaro D, Hernández M, Esteve T, Hoorfar J, Pla M (2003) A rapid and direct real time PCR-based method for identification of Salmonella spp. J Microbiol Methods 54:381–390

    Article  Google Scholar 

  • Rodríguez-Lázaro D, Hernández M, Scortti M, Esteve T, Vázquez-Boland JA, Pla M (2004a) Quantitative detection of Listeria monocytogenes and Listeria innocua by real-time PCR: assessment of hly, iap, and lin02483 targets and AmpliFluor technology. Appl Environ Microbiol 70:1366–1377

    Article  Google Scholar 

  • Rodríguez-Lázaro D, Hernández M, Pla M (2004b) Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay. FEMS Microbiol Lett 233:257–267

    Article  Google Scholar 

  • Rodríguez-Lázaro D, Pla M, Scortti M, Monzó HJ, Vázquez-Boland JA (2005a) A novel real-time PCR for Listeria monocytogenes that monitors analytical performance via an internal amplification control. Appl Environ Microbiol 71:9008–9012

    Article  Google Scholar 

  • Rodríguez-Lázaro D, D’Agostino M, Herrewegh A, Pla M, Cook N, Ikonomopoulos J (2005b) Real-time PCR-based methods for quantitative detection of Mycobacterium avium subsp. paratuberculosis in water and milk. Int J Food Microbiol 101:93–104

    Article  Google Scholar 

  • Rodríguez-Lázaro D, Lewis DA, Ocampo-Sosa AA, Fogarty U, Makrai L, Navas J, Scortti M, Hernández M, Vázquez-Boland JA (2006) An internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi. Appl Environ Microbiol 72:4256–4263

    Article  Google Scholar 

  • Rodríguez-Lázaro D, López-Enríquez L, Hernandez M (2010) smcL as a novel diagnostic marker for quantitative detection of Listeria ivanovii in biological samples. J Appl Microbiol 109:863–872. doi:10.1111/j.1365-2672.2010.04712.x

  • Sangari FJ, Agüero J, García-Lobo JM (2000) The genes for erythritol catabolism are organised as an inducible operon in Brucella abortus. Microbiol 146:487–495

    Article  CAS  Google Scholar 

  • Smith H, Williams AE, Pearce JH, Keppie J, Harris-Smith PW, Fitz-George RB, Witt K (1962) Foetal erythritol: a cause of the localization of Brucella abortus in bovine contagious abortion. Nature 193:47–49

    Article  CAS  Google Scholar 

  • Trapar MK, Young EJ (1986) Urban outbreak of goat cheese brucellosis. Pediatr Infect Dis 5:640–643

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Microbiology Division, Department of Biotechnology and Food Science, University of Burgos, Plaza Misael Bañuelos s/n, Burgos, Spain

    David Rodríguez-Lázaro

  2. Laboratory of Molecular Biology and Microbiology, Instituto Tecnológico Agrario de Castilla y León (ITACyL), Carretera de Burgos, km. 119, 47071, Valladolid, Spain

    Lorena López-Enríquez & Marta Hernández

  3. Microbiology Unit, University Hospital “Marqués de Valdecilla”, Santander, Spain

    Alain A. Ocampo-Sosa

  4. Centro de Investigación y Tecnología Agroalimentaria de Aragón (CITAA), Gobierno de Aragón, Zaragoza, Spain

    Pilar Muñoz, José María Blasco & Clara Marín

  5. Departamento de Ingeniería Agrícola y Forestal, Tecnología de los Alimentos, E.T.S. Ingenierías Agrarias, Universidad de Valladolid, 34004, Palencia, Spain

    Marta Hernández

Authors
  1. David Rodríguez-Lázaro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lorena López-Enríquez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alain A. Ocampo-Sosa
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pilar Muñoz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. José María Blasco
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Clara Marín
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Marta Hernández
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to David Rodríguez-Lázaro or Marta Hernández.

Ethics declarations

Funding

This work was supported by the European Union’s Marie-Curie mobility program (FP7-PEOPLE-2007-2-2-ERG ref. 209050; FoodPath). L.L.-E. received a Ph.D. studentship from the INIA.

Conflict of Interest

David Rodríguez-Lázaro declares that he has no conflict of interest. Lorena López-Enríquez declares that she has no conflict of interest. Alain A. Ocampo- Sosa declares that he has no conflict of interest. Pilar Muñoz declares that he has no conflict of interest. José María Blasco declares that he has no conflict of interest. Clara Marín declares that she has no conflict of interest. Marta Hernández declares that she has no conflict of interest.

Ethical Approval

This article does not contain any studies with human participants.

Informed Consent

Not applicable

Electronic Supplementary Material

ESM 1

(DOCX 59 kb)

ESM 2

(DOCX 46 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rodríguez-Lázaro, D., López-Enríquez, L., Ocampo-Sosa, A.A. et al. Evaluation of eryC as a Molecular Marker for the Quantitative Detection of Brucella Spp. by Real-Time PCR in Food Samples. Food Anal. Methods 10, 1148–1155 (2017). https://doi.org/10.1007/s12161-017-0822-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12161-017-0822-5

Keywords

Search

Navigation