Real time PCR assay for differentiation of Brucella abortus and Brucella melitensis
 

DOI: 10.18805/ijar.v0iOF.8464    | Article Id: B-3349 | Page : 1037-1042
Citation :- Real time PCR assay for differentiation of Brucella abortus and Brucella melitensis .Indian Journal Of Animal Research.2018.(52):1037-1042

Vinay Kumar, Sushila Maan, Aman Kumar, Kanisht Batra, Deepika Chaudhary, Anita Dalal, Akhil K. Gupta, Nitish Bansal, Nancy Sheoran, and N.S. Maan

sushilamaan105@gmail.com
Address :

Department of Animal Biotechnology, College of Veterinary Sciences, Lala Lajpat Rai University of Veterinary and Animal Sciences, Hisar -125 004, Haryana India

Submitted Date : 5-12-2016
Accepted Date : 9-03-2017

Abstract

Brucellosis is one of the zoonotic diseases of major concern and can cause huge economic losses to livestock industry. Serological tests and bacterial isolation are considered as the gold standard assay for diagnosis of Brucella spp. but they are time-consuming, hazardous and lack specificity. To control and eradicate a disease, a confirmatory diagnostic method which is sensitive, quick and specific is the foremost requirement. Therefore in this study, we evaluated the performances of two newly designed TaqMan real-time PCR assays targeting the BruAB_0168 gene and BMEII0466 gene for Brucella abortus and Brucella melitensis (respectively). Both the assays were found to be highly specific in differentiation of respective species. Both the assays can detect as low as 0.02 fg of DNA and there was no detectable difference found in sensitivity of these two tests. R2 value and efficiency of these tests ranged from 0.992 - 0.998 and 100- 106%, respectively showing that these assays are highly efficient.  Compared to conventional PCR assays these qPCR assays were 100 times higher sensitive. In conclusion, the present study showed that the developed real-time PCR assays are more sensitive, specific, have high reproducibility and repeatability and are faster than serological and conventional PCR methods for differentiation of Brucella abortus and Brucella melitensis.

Keywords

Brucella abortus Brucella melitensis PCR assays Zoonotic diseases

References

  1. Baily, G.G., Krahn J.B., Drasar B.S. and Stoker, N.G. (1992). Detection of Brucella melitensis and Brucella abortus by DNA amplification. J. Trop. Med. Hygiene 95: 271-275.
  2. Bounaadja, L., Albert, D., Chenais, B., Henault, S., Zygmunt, M.S. and Poliak, S. (2009). Real-time PCR for identification of Brucella spp.: a comparative study of IS711, bcsp31 and per target genes. Vet. Microbiol. 137: 156-164.
  3. Bricker, B.J. and Halling S.M. (1995). Enhancement of the Brucella AMOS PCR assay for differentiation of Brucellaabortus vaccine strains S19 and RB51. J. Clin. Microbiol. 33: 1640-1642.
  4. Da Costa, M., Guillou, J.P., Garin-Bastuji, B., Thiébaud, M., and Dubray G. (1996). Specificity of six gene sequences for the detection of the genus Brucella by DNA amplification. J. Appl. Bacteriol. 81: 267-275.
  5. Garin-Bastuji, B., Hummel, N., Gerbier, G., Cau, C., Pouillot, R., Da Costa, M. and Fontaine, J.J. (1999). Non specific serological reactions in the diagnosis of bovine brucellosis: experimental oral infection of cattle with repeated doses of Yersinia enterocolitica O: 9. Vet. Microbiol. 66: 223-233.
  6. Gemechu, M.Y., Gill, J.P., Arora, A.K., Ghatak, K.S. and Singh, D.K. (2011). PCR assay for rapid diagnosis and its role in prevention of human brucellosis in Punjab, India. Int. J. Prev. Med. 2(3): 170-177.
  7. Hinic, V., Brodard, I., Thomann, A., Cvetnic, Z., Makaya, P.V. and Frey, J. (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.
  8. Leal-Klevezas, D.S., Martinez-Vazquez, I.O., Garcia-Cantu, J., Lopezmerino, A. and Martinez-Soriano, J.P. (2000). Use of polymerase chain reaction to detect Brucella abortus biovar 1 in infected goats. Vet. Microbiol. 75: 91-97. 
  9. Newby, D.T., Hadfield, T.L. and Roberto, F.F. (2003). Real-time PCR detection of Brucella abortus: a comparative study of SYBR green I, 5’-exonuclease and hybridization probe assays. Appl. Environ. Microbiol. 69: 4753–-4759.
  10. Nicoletti, P. (1980). The epidemiology of bovine brucellosis. Adv. Vet. Sci. Comp. Med. 24: 69-98.
  11. Nielsen, K., Smith, P., Widdison, J., Gall, D., Kelly, L., Kelly, W. and Nicoletti, P. (2004). Serological relationship between cattle exposed to Brucella abortus, Yersinia enterocolitica O: 9 and Escherichia coli O157: H7. Vet. Microbiol. 100: 25-30.
  12. Ocampo-Sosa, A.A., Agüero-Balbin, J. and Garcia-Lobo, J.M. (2005). Development of a new PCR assay to identify Brucella abortus biovars 5, 6 and 9 and the new subgroup 3b of biovar 3. Vet. Microbiol. 110: 41-51.
  13. Otlu, S., Sahin, M., Ataba, H.I. and Unver, A. (2008). Serological investigations of brucellosis in cattle, farmers and veterinarians in the Kars District of Turkey. Acta Veterinaria Brno. 77: 117-121.
  14. Probert, W.S., Schrader, K.N., Khuong, N.Y., Bystrom, S.L. and Graves, M.H. (2004). Real-time multiplex PCR assay for detection of Brucella spp., B.abortus, and B.melitensis. J. Clin. Microbiol. 42: 1290-1293.
  15. Queipo-ortuno, M.I., Colmenero, J.D., Reguera, J.M., Garcia-Ordonez, M.A., Pachon, M.E. and Gonzalez, M. (2005). Rapid diagnosis of human brucellosis by SYBR Green I-based real-time PCR assay and melting curve analysis in serum samples. Clin. Microbiol. Infect. 11: 713-718.
  16. Redkar, R., Rose, S., Bricker, B. and DelVecchio V. (2001). Real-time detection of Brucella abortus, Brucella melitensis and Brucella suis. Mol.Cell. Probes. 15: 43-52.
  17. Rijpens, N.P., Jannes, G., Van Asbroeck, M., Rossau, R. and Herman L.M.F. (1996). Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probe. Appl. Environ. Microbiol. 62:1683-1688.
  18. Romero, C., Gamazo, C., Pardo, M. and Lopez-Goni, I. (1995). Specific detection of Brucella DNA by PCR. J. Clin. Microbiol. 33: 615-617.
  19. Sambrook, J. and Russell, D.W. (2001). Molecular cloning: A laboratory manual. 3rd edn. New York, Cold spring harbor Laboratory Press.
  20. Singh, B.B., Dhand, N.K. and Gill, J.P.S. (2015). Economic losses occurring due to brucellosis in Indian livestock populations. Prev. Vet. Med. 119(3-4): 211-215.
  21. Surucuoglu, S., El, S., Ural, S., Gazi, H., Kurutepe, S., Taskiran, P. and Yurtsever, S.G. (2009). Evaluation of real-time PCR method for rapid diagnosis of brucellosis with different clinical manifestations. Pol. J. Microbiol. 58: 15-19.
  22. Verger, J.M., Grimont, F., Grimont, P.A.D. and Grayon, M. (1985). Brucella, a monospecific genus as shown by deoxyribonucleic acid hybridization. Int. J. Syst. Bacteriol. 35: 292-295.
  23. Zvizdic, S., Cengic, D., Bratic, M., Mehanic, S., Pinjo, F. and Hamzic, S., (2006). Brucella melitensis review of the human infection case. Bosn. J. Basic. Med. Sci.6: 15-18.
     

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