Comparison of different PCR methods for detection of Brucella spp. in human blood samples

• Autorzy: Al-Ajlan H. , Ibrahim A.S.S. , Al-Salamah A.A.
• Języki publikacji: EN

Abstrakty

EN

For detection of Brucella species by PCR four DNA extraction methods and four targets were compared using pure culture of Brucella melitensis and the best conditions were applied in clinical samples. It was found that the MagNA Pure LC method was the most efficient and sensitivemethod showing a positive PCR reaction with DNA extracted from as low as 25 and 100 CFU suspended in one ml blood and one ml water, respectively. Detection of Brucella spp. by conventional PCR was investigated using four different targets. The results indicated that The B4-B5 amplification method was the most sensitive one as it could amplify DNA extracted from as a low as 25 and 100 CFU/ml suspended in one ml water and blood, respectively. Furthermore real-time PCR was able to detect Brucella using DNA extracted from as low as 50 CFU/ml blood and 15 CFU/ml water, respectively. The best and optimum detection conditions were applied to the clinical samples. Evaluation of conventional PCR assays on blood specimens confirmed 72% of the results obtained by conventional blood culture methods with a specificity of 95%, while serum samples had a sensitivity of 54% and specificity of 100%. Real-time PCR was generally found to be more sensitive and specific for detecting Brucella spp. in blood and serum samples compared to conventional PCR. The real-time PCR done on blood specimens confirmed 77.5% of the results obtained by conventional blood culture methods with specificity of 100%, while 60% of serum samples were found to be positive with specificity of 100%. These results suggest that serum and blood analysis by conventional and real time PCR is a convenient and safe method for rapid and accurate diagnosis of brucellosis.

Słowa kluczowe

EN

Brucella; brucellosis; detection; polymerase chain reaction; blood sample; man; zoonotic disease; health problem; economic problem; DNA extraction; Brucella melitensis; diagnosis

• Wydawca: -
• Czasopismo: Polish Journal of Microbiology
• Rocznik: 2011
• Tom: 60
• Numer: 1
• Opis fizyczny: p.27-33,fig.,ref.

Twórcy

autor

Al-Ajlan H.

• Medical Unit, Department of Botany and Microbiology, Faculty of Science, King Saud University, P.O. 2454, Riyadh 11451, Saudi Arabia

autor

Ibrahim A.S.S.

autor

Al-Salamah A.A.

Bibliografia

• Abdoel T., I.T. Dias, R. Cardoso and H.L. Smit. 2008. Simple and rapid field tests for brucellosis in livestock. Vet. Microbiol. 130: 312-319.
• Baddour M.M. and D.H. Alkhalifa. 2008. Evaluation of three polymerase chain reaction techniques for detection of Brucella DNA in peripheral human blood. Can. J. Microbiol. 54: 352-357.
• Bogdanovich T., M. Skurnik, P.S. Lubeck, P. Ahrens and J. Hoorfar. 2004. Validated 5' nuclease PCR assay for rapid identification of the genus Brucella. J. Clin. Microbiol. 42: 2261-2263.
• Bricker B.J. 2002. PCR as a diagnostic tool for brucellosis. Vet. Microbiol. 90: 435-446.
• Cutler S.J., A.M. Whatmore and N.J. Commander. 2005. Brucellosis - new aspects of an old disease. J. Appl. Microbiol. 98: 1270-128.1
• Diaz R. and I. Moriyo. 1989. Laboratory techniques in the diagnosis of human brucellosis. In: Brucellosis: Clinical and Laboratory Aspects (Young, E.J. and Corbel, M.J., Eds.), pp. 73-83. CRC Press, Boca Raton, FL.
• Elfaki M.G., T. Uz-Zamana, A.A. Al-Hokailb and S.M. Nakeeba. 2005. Detection of brucella DNA in sera from patients with brucellosis by polymerase chain reaction. Diagnos. Microbiol. and Infect. Dis. 53: 1-7.
• Godfroid J. 2002. Brucellosis in Wildlife. Rev. Sci. Tech. Int. Epiz. 21: 277-286.
• Hinić V., I. Brodard, A. Thomann, Ž. Cvetnić, P.V. Makaya, J. Frey and C. Abril. 2008. Novel identification and differentiation of Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae suitable for both conventional and real-time PCR systems. J. Microbiol. Methods. 75: 375-378.
• Mattar G.M., I.A. Khneisser and A.M. Abdelnoor. 1996. Rapid laboratory confirmation of human brucellosis by PCR analysis of a target sequence on the 31-Kilodalton Brucella antigen DNA. J. Clin. Microbiol. 34: 477-478.
• Navarro E., J. Escribano, J.A. Fernandez and J. Solera. 2002. Comparison of three different PCR methods for detection of Brucella spp. in human blood samples. FEMS Immun. Med. Microbiol. 34: 147-151.
• Navarro E., J.A. Fernandez and J. Solera. 1999. PCR assay for diagnosis of human brucellosis. J. Clin. Microbiol. 37: 1654-1655.
• Newby D.T., T.L. Hadfield and F.F Roberto. 2003. Real-time PCR detection of Brucella abortus: a comparative study of SYBR Green I, 5'-exonuclase, and hybridization probe assays. Appl. Environ. Microbiol. 69: 4753-4759.
• Probert W.S., K.N. Schrader, N.Y. Khuong, S.L. Bystrom and M.H. Graves. 2004. Real time multiplex PCR assay for detection of Brucella spp., B. abortus, and B. melitensis. J. Clin. Microbiol. 42: 1290-1293.
• Redkar R., S. Rose, B. Bricker and V. Del Vecchio. 2001. Realtime detection of Brucella abortus, Brucella melitensis and Brucella suis. Mol. Cell. Prob. 15: 43 52.
• Sauret J.M. and N. Villissova. 2002. Human brucellosis. J. Am. Board. Pract. 15: 401-406.
• Yagupsky P. 1999. Detection of Brucella in blood cultures. J. Clin. Microbiol. 37: 3437-3342
• Yugupsky P. 2004. Use of the BACTEC MYCO/F LYTIC medium for detection of Brucella melitensis bacteremia. J. Clin. Microbiol. 42: 2207-2208.
• Zerva L., K. Bourantas, S. Mitka, A. Kansouzidou and N.J. Legakis. 2001. Serum is the preferred clinical specimen for diagnosis of human brucellosis by PCR. J. Clin. Microbiol. 39: 1661-1664.