Comparison of duplex polymerase chain reaction and Rose Bengal test for diagnosis of brucella abortus and brucella melitensis

Detection of brucellosis

Authors

Keywords:

Brucellosis, Rose Bengal, Culture

Abstract

BACKGROUND: Diagnosis of brucellosis requires a rapid and accurate method such as the polymerase chain reaction (PCR). The purpose of this study was simultaneous detection of Brucella abortus (B. abortus) and Brucella melitensis (B. melitensis) in serum samples using the duplex PCR technique and then comparing the results using the Rose Bengal test (RBT).

METHODS: In this comparative-descriptive study, 100 serum samples were collected from a veterinary station located in Shahriar City, Iran. Moreover, the monoplex-PCR of B. abortus and B. melitensis and duplex-PCR for both agents was optimized. The limit of detection (LOD) and specificity test were also checked. Besides, deoxyribonucleic acids (DNAs) were extracted from the serum samples by the DNA extraction solution (DNG-plus) technique. The PCR product was cloned in pTZ57R plasmid by T/A cloning.

RESULTS: B. abortus (494bp) and B. melitensis (733bp) amplicons were observed in 1.5% gel electrophoresis. The LOD of the monoplex-PCR test for both of the agents was 100 genomes per reaction. Additionally, 40 out of 100 samples were positive for RBT, out of them, 35 samples were positive with duplex-PCR, 31 samples were positive for B. abortus, and 4 for B. melitensis; moreover, 20 samples were positive with duplex PCR from 60 negative RBT. From this number, 17 samples of B. abortus and 3 samples of B.melitensis were detected.

CONCLUSION: The number of positive samples by duplex-PCR was more than the RBT; therefore, we can assert duplex-PCR for confirming the RBT results.

References

Hekmatimoghaddam S, Sadeh M, Khalili MB, Mollaabedin M, Sazmand A. Comparison of PCR, Wright agglutination test and blood culture for diagnosis of brucellosis in suspected patients. Pak J Biol Sci. 2013; 16(22): 1589-92.

Rubach MP, Halliday JE, Cleaveland S, Crump JA. Brucellosis in low-income and middle-income countries. Curr Opin Infect Dis. 2013; 26(5): 404-12.

Zeinali M, Doosti S, Amiri B, Gouya MM, Godwin GN. Trends in the Epidemiology of Brucellosis Cases in Iran during the Last Decade. Iran J Public Health. 2022; 51(12): 2791-8.

Moreno E, Blasco JM, Letesson JJ, Gorvel JP, Moriyón I. Pathogenicity and Its Implications in Taxonomy: The Brucella and Ochrobactrum Case. Pathogens. 2022; 11(3).

Tuon FF, Gondolfo RB, Cerchiari N. Human-to-human transmission of Brucella - a systematic review. Trop Med Int Health. 2017; 22(5): 539-46.

Aune K, Rhyan JC, Russell R, Roffe TJ, Corso B. Environmental persistence of Brucella abortus in the Greater Yellowstone Area. The Journal of Wildlife Management. 2012; 76(2): 253-61.

Jiang W, Chen J, Li Q, Jiang L, Huang Y, Lan Y, et al. Epidemiological characteristics, clinical manifestations and laboratory findings in 850 patients with brucellosis in Heilongjiang Province, China. BMC Infect Dis. 2019; 19(1): 439.

Dadar M, Shahali Y, Whatmore AM. Human brucellosis caused by raw dairy products: A review on the occurrence, major risk factors and prevention. Int J Food Microbiol. 2019; 292: 39-47.

Estagnasié C, Surgers L, Berdugo K, Monnier-Cholley L, Schmidt M, Meynard JL, et al. Recurrence of brucellosis on breast implants. Infect Dis Now. 2023; 53(2): 104644.

Wensel CR, Pluznick JL, Salzberg SL, Sears CL. Next-generation sequencing: insights to advance clinical investigations of the microbiome. J Clin Invest. 2022; 132(7).

Dal T, Kara SS, Cikman A, Balkan CE, Acıkgoz ZC, Zeybek H, et al. Comparison of multiplex real-time polymerase chain reaction with serological tests and culture for diagnosing human brucellosis. J Infect Public Health. 2019; 12(3): 337-42.

Ferone M, Gowen A, Fanning S, Scannell AGM. Microbial detection and identification methods: Bench top assays to omics approaches. Compr Rev Food Sci Food Saf. 2020; 19(6): 3106-29.

Al-shemmari I. Comparative study between conventional and molecular tests to detect the incidence of brucellosis in cattle and buffaloes in Babylon and Karbala provinces. Scientific Journal of Medical Research. 2018; 02: 07-13.

Yagupsky P, Morata P, Colmenero JD. Laboratory Diagnosis of Human Brucellosis. Clin Microbiol Rev. 2019; 33(1).

Saadat S, Mardaneh J, Ahouran M, Mohammadzadeh A, Ardebili A, Yousefi M, et al. Diagnosis of Cattle Brucellosis by PCR and Serological Methods: Comparison of Diagnostic Tests. Biomedical and Pharmacology Journal. 2017; 10(2): 881-8.

Sotolongo-Rodríguez D, Gomez-Flores R, Navarro-Soto MC, Arellano-Reynoso B, Tamez-Guerra P, Ramírez-Pfeiffer C. Evaluation of the Fluorescence Polarization Assay for the Diagnosis of Brucellosis in Goat Milk. Vet Sci. 2022; 9(6): 303.

Queipo-Ortuño MI, Colmenero JD, Reguera JM, García-Ordoñez MA, Pachón ME, Gonzalez M, et al. Rapid diagnosis of human brucellosis by SYBR Green I-based real-time PCR assay and melting curve analysis in serum samples. Clin Microbiol Infect. 2005; 11(9): 713-8.

Soleimani M, Shams S, Majidzadeh AK. Developing a real-time quantitative loop-mediated isothermal amplification assay as a rapid and accurate method for detection of Brucellosis. J Appl Microbiol. 2013; 115(3): 828-34.

Waringa NMA, Waiboci LW, Bebora L, Kinyanjui PW, Kosgei P, Kiambi S, et al. Human brucellosis in Baringo County, Kenya: Evaluating the diagnostic kits used and identifying infecting Brucella species. PLoS One. 2023; 18(1): e0269831.

Unver A, Erdogan H, Atabay H, Sahin M, Celebi O. Isolation, identification, and molecular characterization of Brucella melitensis from aborted sheep fetuses in Kars, Turkey. Revue de Medecine Veterinaire. 2006; 157(1): 42.

Ewalt DR, Bricker BJ. Validation of the abbreviated Brucella AMOS PCR as a rapid screening method for differentiation of Brucella abortus field strain isolates and the vaccine strains, 19 and RB51. J Clin Microbiol. 2000; 38(8): 3085-6.

Dean AS, Crump L, Greter H, Schelling E, Zinsstag J. Global burden of human brucellosis: a systematic review of disease frequency. PLoS Negl Trop Dis. 2012; 6(10): e1865.

Navarro-Martínez A, Navarro E, Castaño MJ, Solera J. Rapid diagnosis of human brucellosis by quantitative real-time PCR: a case report of brucellar spondylitis. J Clin Microbiol. 2008; 46(1): 385-7.

Díaz R, Casanova A, Ariza J, Moriyón I. The Rose Bengal Test in human brucellosis: a neglected test for the diagnosis of a neglected disease. PLoS Negl Trop Dis. 2011; 5(4): e950.

Yohannes M, Gill JP, Ghatak S, Singh DK, Tolosa T. Comparative evaluation of the Rose Bengal plate test, standard tube agglutination test and complement fixation test for the diagnosis of human brucellosis. Rev Sci Tech. 2012; 31(3): 979-84.

Downloads

Published

2025-02-04

How to Cite

1.
Bahrami S, Shakib P, Zolfaghari MR, Shahhosseini MH, Ahmadi S. Comparison of duplex polymerase chain reaction and Rose Bengal test for diagnosis of brucella abortus and brucella melitensis: Detection of brucellosis. Chron Dis J. 2025;13(1):26–32.

Issue

Section

Original Article(s)

Most read articles by the same author(s)