Evaluation of Different Primers for Detection of Brucella by Using PCR Method
Keywords:
Brucellosis, Diagnosis, Polymerase chain reaction, IS711geneAbstract
Introduction: Brucellosis is a worldwide zoonosis and a significant cause of loss of health in humans and animals. Traditionally, classic diagnosis is carried out by isolation of Brucella, which is time-consuming, technically challenging and potentially dangerous. The aim of this study was to expand a molecular test that would be used for the develop detection of Brucella in a single reaction with high sensitivity and specificity, by targeting IS711element.
Methods: This study was carried out from 2015 to 2016 at the Ayatolla Rohani hospital in Babol, Iran. The present study was designed to develop PCR assay, based on IS711 gene for rapid diagnosis of Brucella spp. and immediate detection of Brucella, with high sensitivity and specificity. Four pairs of oligo-nucleotide primers with sizes of 547, 403, 291 and 127bp respectively, were planned to exclusively amplify the targeted genes of Brucella species.
Results: Our results show that, five PCR primers set up, would be helpful in amplifying the DNAs from the genus Brucella with high specificity and sensitivity so it can be 12 fg, for Brucella species to provide a valuable tool for diagnosis.
Conclusion: This method can be more useful than serological and biochemical tests and in addition, this reduces the number of required tests more rapidly and economically.
References
Christopher S, Umapathy BL, Ravikumar KL. Brucellosis: review on the recent trends in pathogenicity and
laboratory diagnosis. J Lab Physicians. 2010; 2(2): 55-60. doi: 10.4103/0974-2727.72149. PMID:
, PMCID: PMC3040083.
De Santis R, Ciammaruconi A, Faggioni G, Fillo S, Gentile B, Di Giannatale E, et al. High throughput
MLVA-16 typing for Brucella based on the microfluidics technology. BMC Microbiol. 2011; 11: 60. doi:
1186/1471-2180-11-60. PMID: 21435217, PMCID: PMC3078841.
Franco MP, Mulder M, Gilman RH, Smits HL. Human brucellosis. Lancet Infect Dis. 2007; 7(12): 775-86.
doi: 10.1016/S1473-3099(07)70286-4. PMID: 18045560.
Scholz HC, Kämpfer P, Cloeckaert A. Brucella: relationship to other Alphaproteobacteria, current
taxonomy and the emergence of new species. Brucella: Molecular Microbiology and Genomics. 2012; 1.
Araj GF. Update on laboratory diagnosis of human brucellosis. Int J Antimicrob Agents. 2010; 36(1): 12-7.
doi: 10.1016/j.ijantimicag.2010.06.014. PMID: 20692128.
Alamian S, Aghaiipoor K, Salehi T, Esmaelizad M, Fasaei B, Etemadi A. Detection of Brucella melitensis
and Brucella abortus strains using a single-stage PCR method. Arch Razi Inst. 2015; 70(1): 51-5.
Owlia MB, Danesh-Ardakani M. Frequency of sacroiliitis among patients with low back pain. Electron
Physician. 2016; 8(3): 2094-100. doi: 10.19082/2094. PMID: 27123217, PMCID: PMC4844474.
Pappas G. The changing Brucella ecology: novel reservoirs, new threats. Int J Antimicrob Agents. 2010;
(1): 8-11. doi: 10.1016/j.ijantimicag.2010.06.013. PMID: 20696557.
Al Dahouk S, Nockler K. Implications of laboratory diagnosis on brucellosis therapy. Expert Rev Anti
Infect Ther. 2011; 9(7): 833-45. doi: 10.1586/eri.11.55. PMID: 21810055.
Roushan MR, Amiri MJ. Update on childhood brucellosis. Recent Pat Antiinfect Drug Discov. 2013; 8(1):
-6. doi: 10.2174/1574891X11308010008. PMID: 22812616.
Hasanjani Roushan MR, Ebrahimpour S, Moulana Z. Different Clinical Presentations of Brucellosis.
Jundishapur J Microbiol. 2016; 9(4): 33765. doi: 10.5812/jjm.33765. PMID: 27284398, PMCID:
PMC4897599.
Nielsen K, Yu W. Serological diagnosis of brucellosis. Prilozi. 2010; 31(1): 65-89. PMID: 20703184.
Pabuccuoglu O, Ecemis T, El S, Coskun A, Akcali S, Sanlidag T. Evaluation of serological tests for
diagnosis of brucellosis. Jpn J Infect Dis. 2011; 64(4): 272-6. PMID: 21788700.
Hasanjani Roushan MR, Moulana Z, Mohseni Afshar Z, Ebrahimpour S. Risk Factors for Relapse of
Human Brucellosis. Glob J Health Sci. 2016; 8(7): 53370. doi: 10.5539/gjhs.v8n7p77. PMID: 26925907.
Navarro E, Escribano J, Fernandez J, Solera J. Comparison of three different PCR methods for detection of
Brucella spp in human blood samples. FEMS Immunol Med Microbiol. 2002; 34(2): 147-51. PMID:
Cloeckaert A, Grayon M, Grepinet O. An IS711 Element Downstream of thebp26 Gene Is a Specific
Marker of Brucella spp. Isolated from Marine Mammals. Clin diagnos lab immunol. 2000; 7(5): 835-9.
PMID: 10973465, PMCID: PMC95966.
Pérez-Sancho M, García-Seco T, Arrogante L, García N, Martinez I, Diez-Guerrier A, et al. Development
and evaluation of an IS711-based loop mediated isothermal amplification method (LAMP) for detection of
Brucella spp. on clinical samples. Res vet sci. 2013; 95(2): 489-94. doi: 10.1016/j.rvsc.2013.05.002.
PMID: 23714043.
Bounaadja L, Albert D, Chénais B, Hénault S, Zygmunt MS, Poliak S, et al. Real-time PCR for
identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes. Vet Microbiol.
; 137(1-2): 156-64. doi: 10.1016/j.vetmic.2008.12.023. PMID: 19200666.
Navarro E, Segura JC, Castaño MJ, Solera J. Use of real-time quantitative polymerase chain reaction to
monitor the evolution of Brucella melitensis DNA load during therapy and post-therapy follow-up in
patients with brucellosis. Clin infect dis. 2006; 42(9): 1266-73. doi: 10.1086/503035. PMID: 16586386.
Truong QL, Yoon BI, Hahn TW. Development of a multiplex PCR to identify Salmonella, Leptospira and
Brucella species in tissue samples. Korean J Vet Res. 2012; 52(2): 75-82.
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. PMID: 21301197.
Da Costa M, Guillou JP, Garin-Bastuji B, Thiébaud M, Dubray G. Specificity of six gene sequences for the
detection of the genus Brucella by DNA amplification. J Appl Bacteriol. 1996; 81(3): 267-75. PMID:
Hansen WL, Bruggeman CA, Wolffs PF. Pre-analytical sample treatment and DNA extraction protocols for
the detection of bacterial pathogens from whole blood. Methods Mol Biol. 2013; 943: 81-90. doi:
1007/978-1-60327-353-4-4. PMID: 23104282.
Al Dahouk S, Sprague LD, Neubauer H. New developments in the diagnostic procedures for zoonotic
brucellosis in humans. Rev Sci Tech. 2013; 32(1): 177-88. PMID: 23837375.
Buyukcangaz E, Sen A, Carli K, Kahya S. Comparison of direct culture versus PCR for the detection of
Brucella in aborted fetuses of cattle and sheep in Turkey. Vet Rec. 2011; 168(16): 430. doi:
1136/vr.c7003. PMID: 21493448.
Huber B, Scholz HC, Lucero N, Busse HJ. Development of a PCR assay for typing and subtyping of
Brucella species. Int J Med Microbiol. 2009; 299(8): 563-73. doi: 10.1016/j.ijmm.2009.05.002. PMID:
Published
Issue
Section
License
Copyright (c) 2020 KNOWLEDGE KINGDOM PUBLISHING
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.