Multiplex PCR Assay for the Simultaneous Detection of the Brucella Genus in Human Whole Blood and Serum

Mohsen Zamanian1
, Elham Jahani2
, Hassan Mahmoudi3, *

1 Department of Pathobiology, Faculty of Veterinary Medicine, Ferdowsi University of Mashhad, Mashhad, Iran
2 Department of Pathobiology, Faculty of Veterinary Medicine, Bahonar University of Kerman, Kerman, Iran
3 Department of Microbiology, Hamadan University of Medical Sciences, Hamadan, Iran

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Creative Commons License
© 2020 Zamanian et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Department of Microbiology, Hamadan University of Medical Sciences, Hamadan, Iran; Tel: +98-9189539458; E-mail:



Brucellosis disease is a serious zoonosis worldwide and only 17 countries have been recognized as free of brucellosis. The World Health Organization has reported that the incidence of brucellosis is 500,000 cases in a year. Multiplex polymerase chain reaction (PCR) is an ideal method for the identification of brucellosis. The most common primers for the diagnosis of Brucella include B4/B5 and F4/R2. The advantages of multiplex PCR include targeting multiple sequences at the same time, and multiple results are produced in a single test run which saves time and the reagents simultaneously. The purpose of this investigation was to extend and optimize a multiplex PCR for the identification of genus Brucella from serum and whole blood samples.


In this experimental and sectional study, blood samples of 25 suspected patients in the acute phase of brucellosis with serum titers higher than 1:80 were collected. Two pairs of specific primers of B4 and B5 the specific gene was amplified. PCR and Multiplex PCR were performed on blood and serum samples.


Among 25 blood samples, 15 cases (60%) and 9 cases (36%) and among 25 serum samples, 23 cases (92%) and 13 cases (52%) were positive for B4/B5 and F4/R2 in PCR, respectively. In multiplex PCR, among 25 blood samples, 5 cases (20%) showed both bands, 11 cases (44%) showed band 222bp, 4 cases (16%) showed band 905bp and 5 cases (20%) showed no bands. Among 25 serum samples, 6 cases (24%) showed both bands, 15 cases (60%) showed band 222bp, 3 cases (12%) showed band 905bp and 1 case (4%) showed no bands.


The results of this study show that this multiplex PCR can be used for the diagnosis of brucellosis with high sensitivity in clinical laboratories routinely and it can serve as an alternative substitution for risky culture method and nonspecific serological methods.

Keywords: Brucella, Multiplex PCR, Whole blood, Serum, Brucellosis, World Health Organization (WHO).