Detection of a specifically amplified DNAfragment in Brucella abortus by arbitrarilyprimed polymerase chain reaction |
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Authors: | AlMomin S. Saleem M. Al-Mutawa Q. Cheema R.A. |
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Affiliation: | (1) Biotechnology Department, Kuwait Institute for Scientific Research, PO Box 24885, 13109 Safat, Kuwait;(2) Department of Animal Health, Public Authority for Agriculture and Fisheries, PO Box 21422, Safat, Kuwait |
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Abstract: | Randomly amplified polymorphic DNA (RAPD) profiles of Brucella and non-Brucella DNA were established after polymerase chain reaction (PCR) amplification. Five arbitrary oligonucleotide primers were screened to generate Brucella-specific DNA fingerprints. The arbitrary primer OPB-01 (5-GTTTCGCTCC-3) produced DNA bands specific to Brucella. Amplification conditions must be optimized for reproductibility. Accordingly, we optimized and established the conditions, which included Mg2+, enzyme (DNA polymerase), primer, template and deoxyribonucleoside triphosphate (dNTP) concentrations as well as the optimum number of thermal cycles to produce OPB-01 directed Brucella DNA fingerprints.The optimized RAPD method can produce a 1.3 kb DNA fragment specific to Brucella. This DNA fragment was common to eight biovars of B. abortus and one biovar of B. melitensis. The fragment was not detected in genetically related species such as Ochrobactrum anthropi and other non-Brucella organisms associated with farm animals. We anticipate the use of this fragment as a possible probe for the detection of Brucella organisms. |
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Keywords: | Arbitrary primers Brucella abortus Brucella melitensis polymerase chain reaction RAPD |
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