Document Type : Original Research


1 Molecular Biology Research Center, Baqiyatallah University of Medical Sciences. Tehran. iran

2 Molecular Biology Research Center , Baqiyatallah University of Medical Sciences. Tehran. iran

3 Dept. of Biology, Tonekabon Branch, Islamic Azad University of Tonekabon, Tonekabon, Iran

4 Applied Microbiology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.

5 Baqiyatallah Hospital, Baqiyatallah University of Medical Sciences. Tehran. iran


Background: Rapid diagnosis and differentiation of Brucella is of high importance due to the side effects of antibiotics for the treatment of brucellosis. This study aimed to identify and compare PCR-ELISA as a more accurate diagnositc test with other common molecular and serological tests. Methods: In this experimental and sectional study, during March 2014 to Sep 2015, 52 blood samples of suspected patients with clinical symptoms of brucellosis were evaluated in medical centers all over Iran with serum titers higher than 1:80. Using two pairs of specific primers of Brucella abortus, B. melitensis and DIG-dUTP, Fragment IS711 (The common gene fragment in B. melitensis and B. abortus) was amplified. DIG-ELISA was performed using specific probes of these 2 species of Brucella and patterns were subsequently analyzed, then positive responses were compared by detecting gel electrophoresis. Results: PCR-ELISA method detected all 28 samples from 52 positive samples. Its sensitivity was 6.0 pg concentration of genomic DNA of Brucella. In gel electrophoresis method, 22 samples of all positive samples were detected. PCR-ELISA was more efficient than PCR and bacterial culture method at P-value Conclusion: PCR-ELISA molecular method is more sensitive than other molecular methods, lack of mutagenic color and also a semi-quantitative ability. This method is more effective and more accurate compared to PCR, serology and culture of bacteria. PCR-ELISA does not have false responses. The limitation of this method is detection of bacteria in the genus compared to Multiplex PCR and Gel Electrophoresis.


Main Subjects

  1. Mirnejad R, Hosseini Doust R, Kachuei R, Mortazavi SM, Khoobdel M, Ahamadi A. Simultaneous detection and differentiates of Brucella abortus and Brucella melitensis by combinatorial PCR. Asian Pac J Trop Med 2012;5(1):24-8.
  2. Mirnejad R, Mohamadi M, Piranfar V, Mortazavi SM, Kachuei R. A duplex PCR for rapid and simultaneous detection of Brucella spp. in human blood samples. Asian Pac J Trop Med 2013;6(6):453-6.
  3. Mohamed Zahidi J, Bee Yong T, Hashim R, Mohd Noor A, Hamzah SH, Ahmad N. Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis. Diagn Microbiol Infect Dis 2015;81(4):227-33.
  4. Piranfar V, Sharif M, Hashemi M, Vahdati AR, Mirnejad R. Detection and discrimination of two Brucella species by multiplex real-time PCR and high-resolution melt analysis curve from human blood and comparison of results using RFLP. Iran J Basic Med Sci 2015;18(9):909-14.
  5. Trangoni MD, Gioffre AK, Ceron Cucchi ME, Caimi KC, Ruybal P, Zumarraga MJ, et al. LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis. Braz J Microbiol 2015;46(2):619-26.
  6. Parlak M, Akbayram S, Dogan M, Tuncer O, Bayram Y, Ceylan N, et al. Clinical manifestations and laboratory findings of 496 children with brucellosis in Van, Turkey. Pediatr Int 2015;57(4):586-9.
  7. Mugizi DR, Muradrasoli S, Boqvist S, Erume J, Nasinyama GW, Waiswa C, et al. Isolation and molecular characterization of Brucella isolates in cattle milk in Uganda. Biomed Res Int 2015;2015:720413.
  8. Coelho AC, Garcia Diez J. Biological Risks and Laboratory-Acquired Infections: A Reality That Cannot be Ignored in Health Biotechnology. Front Bioeng Biotechnol 2015;3:56.
  9. Dieste-Perez L, Blasco JM, de Miguel MJ, Moriyon I, Munoz PM. Diagnostic performance of serological tests for swine brucellosis in the presence of false positive serological reactions. J Microbiol Methods 2015;111:57-63.
  10. Yu WL, Nielsen K. Review of detection of Brucella spp. by polymerase chain reaction. Croat Med J. 2010;51(4):306-13.
  11. Godfroid J, Nielsen K, Saegerman C. Diagnosis of brucellosis in livestock and wildlife. Croat Med J 2010;51(4):296-305.
  12. Romero C, Pardo M, Grillo MJ, Diaz R, Blasco JM, Lopez-Goni I. Evaluation of PCR and indirect enzyme-linked immunosorbent assay on milk samples for diagnosis of brucellosis in dairy cattle. J Clin Microbiol 1995;33(12):3198-200.
  13. Al Dahouk S, Tomaso H, Nockler K, Neubauer H. The detection of Brucella spp. using PCR-ELISA and real-time PCR assays. Clin Lab 2004;50(7-8):387-94.
  14. Coutlee F, Bobo L, Mayur K, Yolken RH, Viscidi RP. Immunodetection of DNA with biotinylated RNA probes: a study of reactivity of a monoclonal antibody to DNA-RNA hybrids. Anal Biochem 1989;181(1):96-105.
  15. Al Dahouk S, Tomaso H, Nockler K, Neubauer H, Frangoulidis D. Laboratory-based diagnosis of brucellosis--a review of the literature. Part I: Techniques for direct detection and identification of Brucella spp. Clin Lab 2003;49(9-10):487-505.
  16. Morata P, Queipo-Ortuno MI, Reguera JM, Garcia-Ordonez MA, Cardenas A, Colmenero JD. Development and evaluation of a PCR-enzyme-linked immunosorbent assay for diagnosis of human brucellosis. J Clin Microbiol 2003;41(1):144-8.
  17. Araj GF, Kattar MM, Fattouh LG, Bajakian KO, Kobeissi SA. Evaluation of the PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays for diagnosis of human brucellosis. Clin Diagn Lab Immunol 2005;12(11):1334-5.
  18. Bricker BJ. PCR as a diagnostic tool for brucellosis. Vet Microbiol. 2002;90(1-4):435-46.
  19. Sohrabi M, Mohabati Mobarez A, Khoramabadi N, Hosseini Doust R, Behmanesh M. Efficient diagnosis and treatment follow-up of human brucellosis by a novel quantitative TaqMan real-time PCR assay: a human clinical survey. J Clin Microbiol 2014;52(12):4239-43.
  20. Nimri LF. Diagnosis of recent and relapsed cases of human brucellosis by PCR assay. BMC Infect Dis 2003;3:5.
  21. Kazemi B, Namin SY, Bandepour M, Kafilzadeh F, Gachkar L, Mahmoudinejad F, et al. Detection of Brucella by peripheral blood PCR and comparison with culture and serological methods in suspected cases. Iran J Public Health 2008;37(4):96-102.
  22. Shapouri R, Mohabati Mobarez A, Ahmadi H, Tabaraie B, Hosseini Doust R, Norozian D. Optimization of Brucella abortus fermenter culture conditions and LPS extraction method for antigen production. Res J Microbiol 2008;3(1):1-8.
  23. Hosseini Doust R, Ahamdi Z, Ahamdi A, Hajia M, Izadi M, Mobarez AM. Detection of Brucella abortus by alkB and IS711 based primers. J Res Med Sci 2007;12(2):62-7.
  24. Kumar S, Tuteja U, Sarika K, Singh D, Kumar A, Kumar O. Rapid multiplex PCR assay for the simultaneous detection of the Brucella Genus, B. abortus, B. melitensis, and B. suis. J Microbiol Biotechnol 2011;21(1):89-92.