Somayeh TAVAKOLI, Setareh SHAHNAZARI, Hosein SAFARI, Maysam MARD-SOLTANI, Bahman KHALESI

In silico design and in vitro development of a highly accurate test to detect Brucella species

Background: Conventional methods of detecting Brucella spp. suffer from technical and biological complications. Besides, newlycharacterized species of the genus Brucella could be neglected by previously designed polymerase chain reaction (PCR) tests. Therefore,a more accurate PCR-based test seems to be imminently needed.Materials and methods: Blood samples were collected from 39 patients diagnosed with brucellosis and 25 healthy controls. Multiplesequence alignments (MSA) were performed on 500 Omp2-related protein and gene sequences. Thereafter, specific primers weredesigned and synthesized for the regions with highest conservancy. The collected samples were assessed by PCR test. To overcome thecross-reactivity issue, PCR thermal program was optimized regarding annealing time and temperature.Results: The MSA results indicated that the N terminus region of the Omp2 protein (DNA 5’ end) is associated with highest conservancy.Primers with highest specificity were designed and synthesized. A two-step PCR reaction was successfully designed and optimized. Thedesirable bands were observed in clinical samples with high accuracy.Conclusion: It should be pointed out that using a precisely designed primer pair would bring about early infection detection, moresuccess to detect all natural variants and higher cost-to-efficacy ratio in comparison to other detection methods.

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1. de Figueiredo P, Ficht TA, Rice-Ficht A, Rossetti CA, Adams LG. Pathogenesis and immunobiology of brucellosis: review of brucella–host interactions. Am J Pathol 2015; 185: 1505-1517.
2. Abbas AK, Lichtman AH, Pillai S. Cellular and Molecular Immunology E-Book: Elsevier Health Sciences; 2014.
3. O’callaghan D, Whatmore AM. Brucella genomics as we enter the multi-genome era. Briefings Funct Genomics 2011; 10: 334-341.
4. Cutler SJ. Brucella: molecular and cellular biology I. LopezGoni and I. Moriyon, Eds. Horizon Bioscience, Wymondham, UK, 2004. ISBN 1-904933-04-1.£ 90.00, 432 pp. [Review of the book Brucella: Molecular and Cellular Biology]. Journal of Antimicrobial Chemotherapy 2005; 56: 443-444.
5. Al Dahouk S, Tomaso H, Nöckler K, Neubauer H, Frangoulidis D. Laboratory-based diagnosis of brucellosis--a review of the literature. Part II: serological tests for brucellosis. Clin Lab 2003; 49: 577-589.
6. Adlimoghadam A, Hedayati M, Siadat S, Ahmadi H, Nejati M, Vandyousefi J, Norouzion D. Optimization of PCR conditions for detection of human brucellosis from human serum samples. Res J Microbiol 2008; 3: 352-358.
7. Bricker BJ. PCR as a diagnostic tool for brucellosis. Vet Microbiol 2002; 90: 435-446.
8. Gee JE, De BK, Levett PN, Whitney AM, Novak RT, Popovic T. Use of 16S rRNA gene sequencing for rapid confirmatory identification of Brucella isolates. J Clin Microbiol 2004; 42: 3649-3654.
9. Rijpens NP, Jannes G, Van Asbroeck M, Rossau R, Herman L. Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes. Appl Environ Microbiol 1996; 62: 1683-1688.
10. Keid L, Soares R, Vieira N, Megid J, Salgado V, Vasconcellos S, Da Costa M, Gregori F, Richtzenhain L. Diagnosis of canine brucellosis: comparison between serological and microbiological tests and a PCR based on primers to 16S-23S rDNA interspacer. Vet Res Commun 2007; 31: 951-965.
11. Khalili S, Zakeri A, Hashemi ZS, Masoumikarimi M, Manesh MRR, Shariatifar N, Sani MJ. Structural analyses of the interactions between the thyme active ingredients and human serum albumin. Turk J Biochem 2017; 42: 459-467.
12. Khalili S, Jahangiri A, Hashemi ZS, Khalesi B, Mardsoltani M, Amani J. Structural pierce into molecular mechanism underlying Clostridium perfringens Epsilon toxin function. Toxicon 2017; 127: 90-99.
13. Khalili S, Rasaee M, Bamdad T. 3D structure of DKK1 indicates its involvement in both canonical and non-canonical Wnt pathways. Mol Biol 2017; 51: 155-166.
14. Khalili S, Mohammadpour H, BAROUGH MS, Kokhaei P. ILP-2 modeling and virtual screening of an FDA-approved library: a possible anticancer therapy. Turk J Med Sci 2016; 46: 1135-1143.
15. Kazemi Moghaddam E, Owlia P, Jahangiri A, Rasooli I, Rahbar MR, Aghajani M. Conserved OprF as a selective immunogen against pseudomonas aeruginosa. Iran J Pathol 2017; 12: 165- 170.
16. Jahangiri A, Rasooli I, Owlia P, Fooladi AAI, Salimian J. Highly conserved exposed immunogenic peptides of Omp34 against Acinetobacter baumannii: an innovative approach. Journal of microbiological methods 2018; 144: 79-85.
17. Mard-Soltani M, Rasaee MJ, Khalili S, Sheikhi A, Hedayati M, Ghaderi-Zefrehi H, Alasvand M. The effect of differentially designed fusion proteins to elicit efficient anti-human thyroid stimulating hormone immune responses. Iran J Allergy Asthma Immunol 2018; 17: 158-170.
18. Khalili S, Rasaee MJ, Mousavi SL, Amani J, Jahangiri A, Borna H. In silico Prediction and in vitro Verification of a Novel MultiEpitope Antigen for HBV Detection. Mol. Genet. Microbiol. 2017; 32: 230-240.
19. Arabestani MR, Fazzeli H, Esfahani BN. Identification of the most common pathogenic bacteria in patients with suspected sepsis by multiplex PCR. J Infect Dev Ctries 2014; 8: 461-468.
20. Diaz R, Moriyón I. Laboratory techniques in the diagnosis of human brucellosis. In: Young EJ, Corbel MJ, editors. Brucellosis: Clinical and Laboratory Aspect. Boca Raton, FL, USA: CRC Press, Inc.; 1989. pp. 73-83.
21. Young E. Especies de brucella. Enfermedades Infecciosas, Principio y Practica 1997 (in Spanish).
22. Fekete A, Bantle J, Halling SM, Sanborn MR. Preliminary development of a diagnostic test for Brucella using polymerase chain reaction. J Appl Microbiol 1990; 69: 216-227.
23. Wang Y, Wang Z, Zhang Y, Bai L, Zhao Y, Liu C, Ma A, Yu H. Polymerase chain reaction–based assays for the diagnosis of human brucellosis. Ann Clin Microbiol Antimicrob 2014; 13: 31.
24. Ficht T, Bearden S, Sowa B, Marquis H. Genetic variation at the omp2 porin locus of the brucellae: species‐specific markers. Mol Microbiol 1990; 4: 1135-4112.
25. Vizcaíno N, Cloeckaert A, Verger J-M, Grayon M, FernándezLago L. DNA polymorphism in the genus Brucella. Microbes Infect 2000; 2: 1089-1100.
26. Leal-Klevezas DS, Martínez-Vázquez IO, Lopez-Merino A, Martínez-Soriano JP. Single-step PCR for detection of Brucella spp. from blood and milk of infected animals. J Clin Microbiol 1995; 33: 3087-3090.
27. Ficht TA, Bearden S, Sowa B, Adams L. A 36-kilodalton Brucella abortus cell envelope protein is encoded by repeated sequences closely linked in the genomic DNA. Infect Immun 1988; 56: 2036-2046.
28. Ficht T, Husseinen H, Derr J, Bearden S. Species-specific sequences at the omp2 locus of Brucella type strains. Int J Syst Evol Microbiol 1996; 46: 329-331.
29. Baddour MM, Alkhalifa DH. Evaluation of three polymerase chain reaction techniques for detection of Brucella DNA in peripheral human blood. Can J Microbiol 2008; 54: 352-357.
30. Romero C, Gamazo C, Pardo M, Lopez-Goñi I. Specific detection of Brucella DNA by PCR. J Clin Microbiol 1995; 33: 615-617.
31. Fox KF, Fox A, Nagpal M, Steinberg P, Heroux K. Identification of Brucella by ribosomal-spacer-region PCR and differentiation of Brucella canis from other Brucella spp. pathogenic for humans by carbohydrate profiles. J Clin Microbiol 1998; 36: 3217-3222.