Comparison of three primer pairs included: novel primers IS711, universal primers B4 - B5 and 16SrRNA in the diagnosis of human brucellosis in suspected patients in Iran
Researcher Bulletin of Medical Sciences,
Vol. 25 No. 1 (2020),
The genus Brucella is a worldwide distributed intracellular bacteria, which infects animals and human. Currently, this zoonosis has been diagnosed by microbiological and serological laboratory tests. Different PCR protocols with various primer pairs and different target genes have been published for the detection of Brucella, but only a few of these primers have been used in human samples. This study aimed to evaluate and compare the sensitivity and specificity of three primer pairs in the PCR technique, each of which separately amplifies three different regions in the Brucella genome, to determine which are more comfortable for the detecting of Brucella DNA in human clinical samples.
49 clinical serum samples were isolated from suspected patients in different cities in Iran from October 2017 to July 2018. The suspected patients with brucellosis-compatible symptoms were checked. These primers amplified 3 distinctive fragments in BCSP 31 gene (B4/B5), Designed IS711 primers, and a sequence of 16SrRNA of Brucella melitensis.
The results showed that the B4/B5 primer pair had the highest sensitivity and specificity for the detection of both positive and negative samples (100%). The designed IS711 primer pair detected 94% of samples, whereas the 16SrRNA primer pair was the least sensitivity, being able to detect only 30.64% of samples.
The specificity of 3 techniques was 100%. The B4/B5 primers were able to detect the smallest number of bacteria 0.05 CFU/reaction whereas IS711 was able to detect 2 CFU/reaction and 16SrRNA was able to detect 2×105 CFU/reaction.
- Brucella melitensis; Human brucellosis; PCR; Blood samples; Serum samples
How to Cite
2. Che Lh, Qi C, Bao Wg, Ji Xf, Liu J, Du N, Gao L, Zhang Ky, Li Yx. Monitoring the course of Brucella infection with qPCR-based detection. International Journal of Infectious Diseases 2019; 89, 66-71.
3. Wanga H, Xua W, Zhub Kj, Zhua Sj, Zhangc Hf, Wanga J, Yanga Y, Shaob Fy, Jiangd Nm, Taod Zy, Jina Hy, Tanga y, Huoa Ll, Donga F, Lie Zh, Dinga H and Liu Zh. Molecular investigation of infection sources and transmission chains of brucellosis in Zhejiang, China. Emerging Microbes & Infections 2020; 9:1, 889-899.
4. Fero E, Juma A, Koni A, Boci J, Kirandjiski T, Connor R, et al. The seroprevalence of brucellosis and molecular characterization of Brucella species circulating in the beef cattle herds in Albania. PLoS ONE 2020; 15(3): e0229741.https://doi. Org/10.1371 /journal. pone.0229741.
5. Li Z, Wang X, Zhu X, Wang M, Cheng H, Li D and Liu ZG .Molecular Characteristics of Brucella Isolates Collected From Humans in Hainan Province, China. Front. Microbiol 2020; 11:452. doi: 10.3389/fmicb.2020.00452.
6. Gupta VK. Nayakwadi S, Kumar A, Gururaj K, Kumar A, Pawaiya RS. Markers for the Molecular Diagnosis of Brucellosis in Animals. Advances in Animal and Veterinary Sciences 2014; 2(3S):31-39 ·
7. Schol AM, Draeger A, Gollner C, Nockler K. Advancement of a multiplex PCR for the differentiation of all currently described Brucella species. J Microbiolo Methods 2010; 80(1): 112-114.
8. Moreno, E. 1992. Evolution of Brucella, p. 198–218. In M. Plommet (ed.), Prevention of brucellosis in the Mediterranean countries. Proceedings of the International Seminar organized by the International Center for Advanced Agronomic Studies. Pudoc Scientiﬁc Publishers, Wageningen.28-30 October 1991 (CIHEAM Publication, No. 1, 1992).
9. Moreno E, Stackebrandt E, Dorsch M, Wolters J, Busch M., and. Mayer H. Brucella abortus 16S rRNA and lipid A reveal a phylogenetic relationship with members of the alpha-2 subdivision of the class Proteobacteria. J. Bacteriol 1990; 172:3569–3576.
10. Al Dahouk S, Tomaso H, Nöckler K, Neubauer H, Frangoulidis D. Laboratory-based diagnosis of brucellosis—a review of the literature. Part 1: Technique for detection and identification of Brucella spp. Clinical Laboratory 2003; 49(9-10):487-505.
11. Alves CJ, Figueiredo SM, Azevedo SS, Clementino IJ, Keid LB, Vasconcellos SA, Batista CSA, Rocha VCM, Higino SS. Detection of Brucella ovis in ovine from Paraíba State, in the Northeast region of Brazil. Brazilian Journal of Microbiology 2010; 41:365-367.
12. Corbel MJ, Stuart FA, and Brewer RA. Observation on serological cross-reaction between smooth Brucella species and organisms of other genera. Rev. Dev. Biol. Stand 1984; 56:341–348.
13. Diaz, R., and Moriyo LN. 1989. Laboratory techniques in the diagnosis of human brucellosis, p. 73–83. In E. J. Young and M. J. Corbel (ed.), Brucellosis: clinical and laboratory aspects. CRC Press, Inc., Boca Raton, Fla.
14. Baily GG, Krahn JB, Drasar BS, and Stoker NG. Detection of Brucella melitensis and Brucella abortus by DNA ampliﬁcation. J. Trop. Med. Hyg 1992; 95:271–275.
15. Fekete A, Bantle JA, Halling SM. Detection of Brucella by polymerase chain reaction in bovine fetal and maternal tissues.J. Vet.Diagn. Invest 1992; 4:79–83.
16. Herman L, Ridder HD. Identiﬁcation of Brucella spp. by using the polymerase chain reaction. Appl. Environ. Microbiol 1992; 58:2099–2101.
17. Shakerian A, Permal D, Rahimi E, Shahjavan A, Khamesipour F. Molecular detection of Brucella melitensis in sheep and goat milk in Iran.Tropical Journal of Pharmaceutical Research 2016; 15 (5): 913-918.
18. Kwok S, Higushii R, Avoiding false positives by PCR. Nature 1989; 339:237-238.
19. Baily GG, Krahn JB, Drasar BS, Stoker NG. Detection of Brucella melitensis and Brucella abortus by DNA amplification. J Trop Med Hyg 1992; 95(4): 271-275.
20. Kiel FW, Khan NJ. Analysis of 506 consecutive positive serologic tests for brucellosis in Saudi Arabia. J. Clin. Microbiol 1987; 25:1384– 1387.
21. Geresu MA, Kassa GM. A review on diagnostic methods of brucellosis. Journal of Veterinary Science and Technology 2016; 7, 323. doi: 10.4172/2157-7579.1000323
22. Ciftci A, Ica tuba, Savasan S. Evaluation of PCR methods for detection of Brucella strains from culture and tissues. Trop Anim Health Prod 2017; 49: 755-763.
23. Queipo-Ortuno MI Colmenero JD, Reguera JM, Garcia-Ordonez MA, Pachon ME, Gonzalez M, Morata P. 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; 11:713-718.
24. Queipo-Ortuno MI, Morata P, Ocon P, Manchado P, Colmenero JD. Rapid diagnosis of human brucellosis by peripheral-blood PCR assay. J Clin Microbiol 1997; 35(11):2927-2930.
25. Navarro E, Fernandez JA, Escribano J, Solera J. PCR assay for diagnosis of human brucellosis. J Clin Microbiol 1999; 37(5):1654-1655.
26. Zerva L, Bourantas K, Mitka S, Kansouzidou A, Legakis NJ. Serum is the preferred clinical specimen for diagnosis of human brucellosis by PCR. J Clin Microbiol 2001; 39:1661-1664.
27. Al-Attas RA, Al-Khalifa M, Al-Qurashi AR, Badawy M, Al-Gualy N. Evaluation of PCR, culture and serology for the diagnosis of acute human brucellosis. Ann Saudi Med 2000; 20:224-228.
28. Al-Ajlan HH, Ibrahim AS, Al-Salamah AA. Comparison of different PCR methods for detection of Brucella spp. in human blood samples. Pol J Microbiol 2011; 60(1):27-33.
29. Khosravi AD, Abassi E, Alavi SM. Isolation of Brucella melitensis and Brucella abortus from brucellosis patients by conventional culture method and polymerase chain reaction technique. Pak J Med Sci 2006; 22(4):396-400.
30. Elfaki M, Uz-Zaman T, Al-Hokail A, Nakeeb S. Detection of Brucella DNA in sera from patients with brucellosis by polymerase chain reaction. Diagn Microbiol Infect Dis 2005; 53:1-7.
31. Garshasbi M, Ramazani A, Sorouri R, Javani S, Moradi S. Molecular detection of Brucella species in patient suspicious of brucellosis from Zanjan, Iran. Brazilian J Microbiol 2014; 45: 533-538.
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